Engineered uridine phosphorylase variant enzymes

ABSTRACT

The present invention provides engineered uridine phosphorylase (UP) enzymes, polypeptides having UP activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing UP enzymes are also provided. The present invention further provides compositions comprising the UP enzymes and methods of using the engineered UP enzymes. The present invention finds particular use in the production of pharmaceutical compounds.

The present application claims priority to U.S. Prov. Pat. Appln. Ser.No. 63/127,431, filed Dec. 18, 2020 and US Prov. Pat. Appln. Ser. No.63/148,324, filed Feb. 11, 2021, both of which are incorporated byreference in their entireties, for all purposes.

FIELD OF THE INVENTION

The present invention provides engineered uridine phosphorylase (UP)enzymes, polypeptides having UP activity, and polynucleotides encodingthese enzymes, as well as vectors and host cells comprising thesepolynucleotides and polypeptides. Methods for producing UP enzymes arealso provided. The present invention further provides compositionscomprising the UP enzymes and methods of using the engineered UPenzymes. The present invention finds particular use in the production ofpharmaceutical compounds.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “CX2-214US2_ST25.txt”, a creation date of Dec. 14, 2021 anda size of 1.84 megabytes. The Sequence Listing filed via EFS-Web is partof the specification and incorporated in its entirety by referenceherein.

BACKGROUND OF THE INVENTION

Increasingly, non-natural nucleoside analogs are being investigated forthe treatment of cancer and viral infections, such as COVID-19.Nucleoside analogs are often potent inhibitors of viral diseases due totheir similarity to natural nucleosides used in the synthesis of DNA.Similarly, nucleoside analogs are known to stimulate the anti-tumorinnate immune response, which is activated by the presence of foreignnucleic acids.

However, production of nucleoside analogs by standard chemical synthetictechniques can pose a challenge due to their chemical complexity.Additionally, standard chemical synthetic techniques often produceundesirable waste products. Substrates and intermediates necessary toproduce nucleoside analogs may not be available or suitable forindustrial process conditions.

There is a need for improved methods to generate non-natural nucleosideanalogs for treatment of cancer and viral infections. Specifically,improved methods of synthesizing substrates and intermediates necessaryfor generating nucleoside analogs under industrial process conditionsare necessary. One approach is to utilize engineered polypeptides withimproved properties to produce substrates and intermediates. Thisbiocatalytic approach has the advantages of reducing chemical waste andincreasing efficiency.

Enzymes belonging to the uridine phosphorylase class of enzymes (EC2.4.2.3) typically catalyze the conversion of uridine and phosphate touracil and alpha-D-ribose 1-phosphate. However, uridine phosphorylases(UP) also catalyze the reverse reaction and may be useful in a varietyof conversions to synthesize non-natural nucleoside analogs. There isneed for improved uridine phosphorylase biocatalysts to produceintermediates and substrates necessary for the efficient production ofnon-natural nucleoside analogs.

SUMMARY OF THE INVENTION

The present invention provides engineered uridine phosphorylase (UP)enzymes, polypeptides having UP activity, and polynucleotides encodingthese enzymes, as well as vectors and host cells comprising thesepolynucleotides and polypeptides. Methods for producing UP enzymes arealso provided. The present invention further provides compositionscomprising the UP enzymes and methods of using the engineered UPenzymes. The present invention finds particular use in the production ofpharmaceutical compounds.

The present invention provides engineered uridine phosphorylasescomprising polypeptide sequences having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NOs: 2, 4, 246, 594, 776, and/or 868, or a functionalfragment thereof, wherein said engineered uridine phosphorylasecomprises a polypeptide comprising at least one substitution orsubstitution set in said polypeptide sequence, and wherein the aminoacid positions of said polypeptide sequence are numbered with referenceto SEQ ID NOs: 2, 4, 246, 594, 776, and/or 868. In some embodiments, thepolypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NOs: 2, and wherein the polypeptide of the engineered uridinephosphorylase comprises at least one substitution or substitution set atone or more positions in said polypeptide sequence selected from 6, 7,9, 14, 14/38/40/146/147/179/235/236, 14/38/86/146/147/235/236/240,14/40, 14/40/86/147/193/236/240, 14/40/136/179/236/240,14/40/146/235/236, 14/40/147/181/193/235, 14/40/235, 14/86/146,14/146/147/181/240, 14/146/236/240, 14/147/193/235/236/240,14/179/181/193/235/240, 14/235/236, 29, 31, 38/40/86/146/147/179/181,38/40/86/147/236/240, 40, 40/43/86/146/240, 40/43/86/147/235,40/43/146/147, 40/43/147/179/236/240, 40/43/147/179/240,40/43/147/236/240, 40/86/146/235/236, 40/86/147/235/236/240,40/86/179/235/240, 40/86/235/236/240, 40/146/147/240, 40/147/240,40/235, 40/235/236/240, 40/236, 40/236/240, 42/235/236,43/86/147/181/240, 43/146/147/235/236/240, 43/146/179/240, 43/147,43/147/179/181, 47, 47/88, 64, 73, 80, 86, 86/136/146/147/179/181,86/136/146/147/179/235/236, 86/147/179/181, 86/235, 86/235/236/240,86/236/240, 86/240, 92, 97, 99, 103, 103/249, 104, 105, 106, 110,146/147, 146/147/235/236, 146/235/240, 146/236/240, 146/240,147/179/181, 147/235/240/249, 157, 167, 179, 179/181, 179/181/193/240,181, 184, 216, 226, 228, 231, 233, 235, 235/236, 236, 236/240, 237, 239,240, and 245, wherein the amino acid positions of said polypeptidesequence are numbered with reference to SEQ ID NOs: 2. In someembodiments, the polypeptide sequence of the engineered uridinephosphorylase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:2,and wherein the polypeptide of the engineered uridine phosphorylasecomprises at least one substitution or substitution set at one or morepositions in said polypeptide sequence selected from 6F, 6G, 6R, 6S, 6W,6Y, 7T, 9M, 14A, 14A/38L/40S/146E/147L/179R/235R/236A,14A/38L/86V/146E/147L/235R/236A/240R, 14A/40D/86V/147M/193L/236A/240R,14A/40D/235R, 14A/40N, 14A/40N/146E/235R/236A,14A/40N/147L/181Q/193L/235R, 14A/40S, 14A/40S/136V/179R/236A/240R,14A/86V/146E, 14A/146E/147L/181Q/240R, 14A/146E/236A/240R,14A/147L/193L/235R/236A/240R, 14A/179R/181Q/193L/235R/240R,14A/235R/236A, 29S, 31T, 38L/40D/86V/146E/147L/179R/181Q,38L/40S/86V/147L/236A/240R, 40D, 40D/86V/147L/235R/236A/240R,40D/86V/235R/236A/240R, 40D/235R, 40N/43F/86V/146E/240R,40N/43F/86V/147L/235R, 40N/43F/146E/147L, 40N/43F/147L/179R/236A/240R,40N/86V/179R/235R/240R, 40S/43F/147L/179R/240R, 40S/43F/147L/236A/240R,40S/86V/146E/235R/236A, 40S/86V/147L/235R/236A/240R, 40S/146E/147L/240R,40S/147L/240R, 40S/235R/236A/240R, 40S/236A, 40S/236A/240R,42I/235R/236A, 43F/86V/147L/181Q/240R, 43F/146E/147L/235R/236A/240R,43F/146E/179R/240R, 43F/147L, 43F/147L/179R/181Q, 47M, 47R, 47V,47V/88A, 64C, 73G, 80G, 801, 80K, 80M, 80S, 80T, 86V,86V/136V/146E/147L/179R/181Q, 86V/136V/146E/147L/179R/235R/236A,86V/147L/179R/181Q, 86V/235R, 86V/235R/236A/240R, 86V/236A/240R,86V/240R, 92V, 97T, 99E, 103G/249V, 103S, 104T, 105M, 106E, 110A, 110C,110S, 146E/147L, 146E/147L/235R/236A, 146E/235R/240R, 146E/236A/240R,146E/240R, 147L/179R/181Q, 147L/235R/240R/249V, 157S, 167S, 179R,179R/181Q, 179R/181Q/193L/240R, 181Q, 184R, 184S, 216M, 226R, 228A,228G, 228K, 228L, 228R, 231I, 231P, 231T, 231W, 231Y, 233S, 235R,235R/236A, 236A, 236A/240R, 237V, 239A, 239K, 239R, 240R, and 245S,wherein the amino acid positions of said polypeptide sequence arenumbered with reference to SEQ ID NO: 2. In some embodiments, thepolypeptide sequence of the engineered uridine phosphorylase has atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to SEQ ID NO:2, and wherein thepolypeptide of the engineered uridine phosphorylase comprises at leastone substitution or substitution set at one or more positions in saidpolypeptide sequence selected from V6F, V6G, V6R, V6S, V6W, V6Y, F7T,L9M, N14A, N14A/M38L/K40S/S146E/I147L/H179R/K235R/Q236A,N14A/M38L/186V/S146E/I147L/K235R/Q236A/H240R,N14A/K40D/186V/I147M/M193L/Q236A/H240R, N14A/K40D/K235R, N14A/K4ON,N14A/K4ON/S146E/K235R/Q236A, N14A/K4ON/I147L/K181Q/M193L/K235R,N14A/K40S, N14A/K40S/C136V/H179R/Q236A/H240R, N14A/I86V/S146E,N14A/S146E/I147L/K181Q/H240R, N14A/S146E/Q236A/H240R,N14A/I147L/M193L/K235R/Q236A/H240R, N14A/H179R/K181Q/M193L/K235R/H240R,N14A/K235R/Q236A, D29S, V31T, M38L/K40D/I86V/S146E/I147L/H179R/K181Q,M38L/K40S/I86V/I147L/Q236A/H240R, K40D,K40D/I86V/I147L/K235R/Q236A/H240R, K40D/I86V/K235R/Q236A/H240R,K40D/K235R, K40N/K43F/I86V/S146E/H240R, K40N/K43F/I86V/I147L/K235R,K40N/K43F/S146E/I147L, K40N/K43F/I147L/H179R/Q236A/H240R,K40N/I86V/H179R/K235R/H240R, K40S/K43F/I147L/H179R/H240R,K40S/K43F/I147L/Q236A/H240R, K40S/186V/S146E/K235R/Q236A,K40S/I86V/I147L/K235R/Q236A/H240R, K40S/S146E/I147L/H240R,K40S/I147L/H240R, K40S/K235R/Q236A/H240R, K40S/Q236A, K40S/Q236A/H240R,V42I/K235R/Q236A, K43F/186V/I147L/K181Q/H240R,K43F/S146E/I147L/K235R/Q236A/H240R, K43F/S146E/H179R/H240R, K43F/I147L,K43F/I147L/H179R/K181Q, H47M, H47R, H47V, H47V/T88A, V64C, S73G, E80G,E80I, E80K, E80M, E80S, E80T, I86V, I86V/C136V/S146E/I147L/H179R/K181Q,I86V/C136V/S146E/I147L/H179R/K235R/Q236A, I86V/I147L/H179R/K181Q,I86V/K235R, I86V/K235R/Q236A/H240R, I86V/Q236A/H240R, I86V/H240R, I92V,A97T, Q99E, N103G/A249V, N103S, V104T, G105M, D106E, T110A, T110C,T110S, S146E/I147L, S146E/I147L/K235R/Q236A, S146E/K235R/H240R,S146E/Q236A/H240R, S146E/H240R, I147L/H179R/K181Q,I147L/K235R/H240R/A249V, A157S, E167S, H179R, H179R/K181Q,H179R/K181Q/M193L/H240R, K181Q, M184R, M184S, V216M, Q226R, I228A,I228G, I228K, I228L, I228R, A231I, A231P, A231T, A231W, A231Y, T233S,K235R, K235R/Q236A, Q236A, Q236A/H240R, T237V, S239A, S239K, S239R,H240R, and V245S, wherein the amino acid positions of said polypeptidesequence are numbered with reference to SEQ ID NO: 2. In someembodiments, the engineered uridine phosphorylase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO:2. In some embodiments, the engineered uridine phosphorylasecomprises a polypeptide sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 2.In some embodiments, the engineered uridine phosphorylase comprises apolypeptide sequence having at least 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 2.

In some embodiments, the present invention provides an engineereduridine phosphorylase having a polypeptide sequence that is at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more sequence identity to SEQ ID NO:4, and wherein thepolypeptide of said engineered uridine phosphorylase comprises at leastone substitution or substitution set at one or more positions in saidpolypeptide sequence selected from 3, 3/9/216, 3/9/216/236, 3/9/235/237,3/31/47/179/181/216, 3/31/47/179/181/237, 3/31/47/179/216,3/31/47/179/216/237, 3/31/179, 3/31/179/181, 3/31/179/181/237,3/31/179/216, 3/31/181/216, 3/31/181/237, 3/47/179/181/216,3/47/179/216/237, 3/47/181, 3/179, 3/179/181, 3/179/181/216,3/179/181/237, 3/179/216, 3/179/216/237, 3/179/237, 3/181/216,3/181/216/237, 3/216/236/240, 9/216/236/237, 9/237, 13, 24, 31, 31/47,31/47/179/181/237, 31/47/179/216, 31/47/181, 31/47/216, 31/179,31/179/181, 31/179/216, 31/181, 31/181/216, 31/181/216/237, 31/181/237,31/216, 31/216/237, 31/236/237/240, 31/237, 33, 46, 47, 47/147/181/231,47/179/181, 47/179/181/216, 47/179/184, 47/179/216, 47/181/216,47/181/216/237, 47/181/231, 47/216, 52, 63, 67, 83, 87/160, 92, 95, 97,99, 100, 101, 105, 106, 108, 111, 137, 151, 152, 155, 159, 160, 170,173, 177, 179, 179/181, 179/181/216, 179/181/216/237, 179/181/231,179/181/241, 179/216, 179/228/231, 179/237, 181, 181/216, 181/216/237,181/237, 183, 185, 188, 189, 191, 201, 216, 216/237, 218, 222, 228, 231,233, 235, 235/237, 236, 236/237/240, 237, 238, 240, 241, and 248,wherein the amino acid positions of said polypeptide sequence arenumbered with reference to SEQ ID NO: 4. In some embodiments, thepresent invention provides an engineered uridine phosphorylase having apolypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 4, and wherein the polypeptide of said engineered uridinephosphorylase comprises at least one substitution or substitution set atone or more positions in said polypeptide sequence selected from 3E,3E/9M/216M, 3E/9M/216M/236A, 3E/9M/235R/237V, 3E/31T/47V/179R/181Q/216M,3E/31T/47V/179R/181Q/237V, 3E/31T/47V/179R/216M,3E/31T/47V/179R/216M/237V, 3E/31T/179R, 3E/31T/179R/181Q,3E/31T/179R/181Q/237V, 3E/31T/179R/216M, 3E/31T/181Q/216M,3E/31T/181Q/237V, 3E/47V/179R/181Q/216M, 3E/47V/179R/216M/237V,3E/47V/181Q, 3E/179R, 3E/179R/181Q, 3E/179R/181Q/216M,3E/179R/181Q/237V, 3E/179R/216M, 3E/179R/216M/237V, 3E/179R/237V,3E/181Q/216M, 3E/181Q/216M/237V, 3E/216M/236A/240R, 9M/216M/236A/237V,9M/237V, 13L, 24L, 24M, 24Q, 31T, 31T/47V, 31T/47V/179R/181Q/237V,31T/47V/179R/216M, 31T/47V/181Q, 31T/47V/216M, 31T/179R, 31T/179R/181Q,31T/179R/216M, 31T/181Q, 31T/181Q/216M, 31T/181Q/216M/237V,31T/181Q/237V, 31T/216M, 31T/216M/237V, 31T/236A/237V/240R, 31T/237V,33L, 33N, 33R, 33V, 33Y, 46Q, 47V, 47V/147L/181Q/231I, 47V/179R/181Q,47V/179R/181Q/216M, 47V/179R/184R, 47V/179R/216M, 47V/181Q/216M,47V/181Q/216M/237V, 47V/181Q/231W, 47V/216M, 47W, 52L, 52W, 63M, 67G,83G, 87H/160C, 92M, 95S, 97S, 99L, 99R, 99V, 100D, 100E, 100G, 100R,100T, 101A, 101T, 101V, 101W, 105S, 106E, 108A, 108M, 108V, 111K, 111R,137G, 151S, 152L, 152S, 152V, 155R, 159A, 159T, 160G, 170A, 173R, 177R,179R, 179R/181Q, 179R/181Q/216M, 179R/181Q/216M/237V, 179R/181Q/231W,179R/181Q/241V, 179R/216M, 179R/228A/231I, 179R/237V, 181Q, 181Q/216M,181Q/216M/237V, 181Q/237V, 183L, 183T, 183W, 185A, 185G, 185H, 185K,185Q, 185R, 185S, 185V, 185W, 188A, 188L, 189L, 189R, 189V, 191F, 191R,201L, 216M, 216M/237V, 218A, 218V, 222L, 222R, 228H, 228L, 228T, 231G,231V, 233A, 233G, 233N, 233S, 235H, 235R/237V, 235S, 236A,236A/237V/240R, 236P, 236S, 236T, 237V, 238G, 238P, 238S, 240G, 241G,241L, 241M, 241P, 241W, and 248T, wherein the amino acid positions ofsaid polypeptide sequence are numbered with reference to SEQ ID NO: 4.In some embodiments, the present invention provides an engineereduridine phosphorylase having a polypeptide sequence that is at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more sequence identity to SEQ ID NO: 4, and wherein thepolypeptide of said engineered uridine phosphorylase comprises at leastone substitution or substitution set at one or more positions in saidpolypeptide sequence selected from K3E, K3E/L9M/V216M,K3E/L9M/V216M/Q236A, K3E/L9M/K235R/T237V,K3E/V31T/H47V/H179R/K181Q/V216M, K3E/V31T/H47V/H179R/K181Q/T237V,K3E/V31T/H47V/H179R/V216M, K3E/V31T/H47V/H179R/V216M/T237V,K3E/V31T/H179R, K3E/V31T/H179R/K181Q, K3E/V31T/H179R/K181Q/T237V,K3E/V31T/H179R/V216M, K3E/V31T/K181Q/V216M, K3E/V31T/K181Q/T237V,K3E/H47V/H179R/K181Q/V216M, K3E/H47V/H179R/V216M/T237V, K3E/H47V/K181Q,K3E/H179R, K3E/H179R/K181Q, K3E/H179R/K181Q/V216M,K3E/H179R/K181Q/T237V, K3E/H179R/V216M, K3E/H179R/V216M/T237V,K3E/H179R/T237V, K3E/K181Q/V216M, K3E/K181Q/V216M/T237V,K3E/V216M/Q236A/H240R, L9M/V216M/Q236A/T237V, L9M/T237V, K13L, V24L,V24M, V24Q, V31T, V31T/H47V, V31T/H47V/H179R/K181Q/T237V,V31T/H47V/H179R/V216M, V31T/H47V/K181Q, V31T/H47V/V216M, V31T/H179R,V31T/H179R/K181Q, V31T/H179R/V216M, V31T/K181Q, V31T/K181Q/V216M,V31T/K181Q/V216M/T237V, V31T/K181Q/T237V, V31T/V216M, V31T/V216M/T237V,V31T/Q236A/T237V/H240R, V31T/T237V, K33L, K33N, K33R, K33V, K33Y, S46Q,H47V, H47V/I147L/K181Q/A231I, H47V/H179R/K181Q, H47V/H179R/K181Q/V216M,H47V/H179R/M184R, H47V/H179R/V216M, H47V/K181Q/V216M,H47V/K181Q/V216M/T237V, H47V/K181Q/A231W, H47V/V216M, H47W, T52L, T52W,I63M, T67G, Q83G, R87H/D160C, I92M, T95S, A97S, Q99L, Q99R, Q99V, P100D,P100E, P100G, P100R, P100T, H101A, H101T, H101V, H101W, G105S, D106E,L108A, L108M, L108V, T111K, T111R, T137G, T151S, H152L, H152S, H152V,V155R, S159A, S159T, D160G, D170A, S173R, V177R, H179R, H179R/K181Q,H179R/K181Q/V216M, H179R/K181Q/V216M/T237V, H179R/K181Q/A231W,H179R/K181Q/A241V, H179R/V216M, H179R/I228A/A231I, H179R/T237V, K181Q,K181Q/V216M, K181Q/V216M/T237V, K181Q/T237V, S183L, S183T, S183W, E185A,E185G, E185H, E185K, E185Q, E185R, E185S, E185V, E185W, Q188A, Q188L,A189L, A189R, A189V, G191F, G191R, T201L, V216M, V216M/T237V, G218A,G218V, N222L, N222R, I228H, I228L, I228T, A231G, A231V, T233A, T233G,T233N, T233S, K235H, K235R/T237V, K235S, Q236A, Q236A/T237V/H240R,Q236P, Q236S, Q236T, T237V, E238G, E238P, E238S, H240G, A241G, A241L,A241M, A241P, A241W, and A248T, wherein the amino acid positions of saidpolypeptide sequence are numbered with reference to SEQ ID NO: 4. Insome embodiments, the engineered uridine phosphorylase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 4. In some embodiments, the engineered uridine phosphorylasecomprises a polypeptide sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 4.In some embodiments, the engineered uridine phosphorylase comprises apolypeptide sequence having at least 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 4.

In some embodiments, the present invention provides an engineereduridine phosphorylase having a polypeptide sequence that is at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more sequence identity to SEQ ID NO: 246, and wherein thepolypeptide of said engineered uridine phosphorylase comprises at leastone substitution or substitution set at one or more positions in saidpolypeptide sequence selected from 3/24/33/47/100/183/185,3/24/33/47/100/216/228/233, 3/24/33/100/183/185/228, 3/24/33/108,3/24/47/100, 3/24/47/100/108/111/160/185/233/241, 3/24/47/108/160/241,3/24/47/160/189, 3/24/47/189/228/233, 3/24/47/228, 3/24/47/228/233,3/24/95/100, 3/24/95/100/160/189/228/241, 3/24/100,3/24/100/160/218/241, 3/24/111, 3/24/111/183/228/233/241,3/24/111/228/233, 3/24/183/185/216, 3/24/189/233, 3/33/47/95/100/241,3/33/47/100/108/189/216/228/233, 3/33/47/100/111/228,3/33/47/100/111/233/241, 3/33/47/100/216, 3/33/47/108/111/233,3/33/47/108/189/233/241, 3/33/160/233, 3/47, 3/47/95/100/108/189/233,3/47/95/100/111/241, 3/47/95/160/189, 3/47/100/108/183/185/189/241,3/47/100/160/185, 3/47/100/185/189/228, 3/47/108/111,3/47/183/189/228/233, 3/47/189, 3/47/228/233, 3/95/100/160/228/233,3/95/100/183/216/228/233, 3/95/100/183/233, 3/95/185/189/216, 3/95/189,3/95/233, 3/160, 3/183/185/189/228/233, 3/183/189/228/233, 3/185,3/185/189, 3/189, 24, 24/33/47, 24/33/47/228/241, 24/33/100/108/241,24/47/95/100, 24/47/95/100/160/228/233/241, 24/47/185/216/218,24/47/216, 24/95/183, 24/100/160/233, 24/160/183/185, 24/189/228/233,33/47/95/100/233, 33/47/95/100/233/241, 33/47/160, 33/47/233,33/100/183/185, 33/100/185/233, 47, 47/100/111/233, 47/100/189,47/100/189/233, 47/108/160/228/241, 47/111, 47/160/185/189/233,47/228/233, 95/100/183, 95/100/189, 95/100/228, 95/100/228/233,95/100/233, 100/160/185, 100/228/233, 108, 108/183/189/233,108/185/216/228/233, 160/233, 228, and 228/233, wherein the amino acidpositions of said polypeptide sequence are numbered with reference toSEQ ID NO: 246. In some embodiments, the present invention provides anengineered uridine phosphorylase having a polypeptide sequence that isat least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more sequence identity to SEQ ID NO: 246, and whereinthe polypeptide of said engineered uridine phosphorylase comprises atleast one substitution or substitution set at one or more positions insaid polypeptide sequence selected from3E/24L/33Y/47V/100E/216M/228L/233S, 3E/24L/33Y/47W/100E/183L/185A,3E/24L/33Y/100E/183L/185A/228L, 3E/24L/33Y/108V, 3E/24L/47V/228L,3E/24L/47V/228L/233S, 3E/24L/47W/100E,3E/24L/47W/100E/108V/111K/160G/185A/233S/241A,3E/24L/47W/108V/160G/241A, 3E/24L/47W/160G/189R,3E/24L/47W/189R/228L/233S, 3E/24L/95S/100E,3E/24L/95S/100E/160G/189R/228L/241A, 3E/24L/100E,3E/24L/100E/160G/218V/241A, 3E/24L/111K,3E/24L/111K/183L/228L/233S/241A, 3E/24L/111K/228L/233S,3E/24L/183L/185A/216M, 3E/24L/189R/233S, 3E/33Y/47V/100E/111K/228L,3E/33Y/47V/100E/111K/233S/241A, 3E/33Y/47V/100E/216M,3E/33Y/47W/95S/100E/241A, 3E/33Y/47W/100E/108V/189R/216M/228L/233S,3E/33Y/47W/108V/111K/233S, 3E/33Y/47W/108V/189R/233S/241A,3E/33Y/160G/233S, 3E/47V/95S/100E/111K/241A, 3E/47V/95S/160G/189R,3E/47V/100E/160G/185A, 3E/47W, 3E/47W/95S/100E/108V/189R/233S,3E/47W/100E/108V/183L/185A/189R/241A, 3E/47W/100E/185A/189R/228L,3E/47W/108V/111K, 3E/47W/183L/189R/228L/233S, 3E/47W/189R,3E/47W/228L/233S, 3E/95S/100E/160G/228L/233S,3E/95S/100E/183L/216M/228L/233S, 3E/95S/100E/183L/233S,3E/95S/185A/189R/216M, 3E/95S/189R, 3E/95S/233S, 3E/160G,3E/183L/185A/189R/228L/233S, 3E/183L/189R/228L/233S, 3E/185A,3E/185A/189R, 3E/189R, 24L, 24L/33Y/47W, 24L/33Y/47W/228L/241A,24L/33Y/100E/108V/241A, 24L/47V/95S/100E,24L/47V/95S/100E/160G/228L/233S/241A, 24L/47V/185A/216M/218V,24L/47V/216M, 24L/95S/183L, 24L/100E/160G/233S, 24L/160G/183L/185A,24L/189R/228L/233S, 33Y/47V/95S/100E/233S, 33Y/47W/95S/100E/233S/241A,33Y/47W/160G, 33Y/47W/233S, 33Y/100E/183L/185A, 33Y/100E/185A/233S, 47V,47V/100E/111K/233S, 47V/100E/189R, 47V/108V/160G/228L/241A, 47V/111K,47V/160G/185A/189R/233S, 47V/228L/233S, 47W/100E/189R/233S,95S/100E/183L, 95S/100E/189R, 95S/100E/228L, 95S/100E/228L/233S,95S/100E/233S, 100E/160G/185A, 100E/228L/233S, 108V,108V/183L/189R/233S, 108V/185A/216M/228L/233S, 160G/233S, 228L, and228L/233S, wherein the amino acid positions of said polypeptide sequenceare numbered with reference to SEQ ID NO: 246. In some embodiments, thepresent invention provides an engineered uridine phosphorylase having apolypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 246, and wherein the polypeptide of said engineered uridinephosphorylase comprises at least one substitution or substitution set atone or more positions in said polypeptide sequence selected fromK3E/V24L/K33Y/H47V/P100E/V216M/I228L/T233S,K3E/V24L/K33Y/H47W/P100E/S183L/E185A,K3E/V24L/K33Y/P100E/S183L/E185A/I228L, K3E/V24L/K33Y/L108V,K3E/V24L/H47V/I228L, K3E/V24L/H47V/I228L/T233S, K3E/V24L/H47W/P100E,K3E/V24L/H47W/P100E/L108V/T111K/D160G/E185A/T233S/M241A,K3E/V24L/H47W/L108V/D160G/M241A, K3E/V24L/H47W/D160G/A189R,K3E/V24L/H47W/A189R/I228L/T233S, K3E/V24L/T95S/P100E,K3E/V24L/T95S/P100E/D160G/A189R/I228L/M241A, K3E/V24L/P100E,K3E/V24L/P100E/D160G/G218V/M241A, K3E/V24L/T111K,K3E/V24L/T111K/S183L/I228L/T233S/M241A, K3E/V24L/T111K/I228L/T233S,K3E/V24L/S183L/E185A/V216M, K3E/V24L/A189R/T233S,K3E/K33Y/H47V/P100E/T111K/I228L, K3E/K33Y/H47V/P100E/T111K/T233S/M241A,K3E/K33Y/H47V/P100E/V216M, K3E/K33Y/H47W/T95S/P100E/M241A,K3E/K33Y/H47W/P100E/L108V/A189R/V216M/I228L/T233S,K3E/K33Y/H47W/L108V/T111K/T233S, K3E/K33Y/H47W/L108V/A189R/T233S/M241A,K3E/K33Y/D160G/T233S, K3E/H47V/T95S/P100E/T111K/M241A,K3E/H47V/T95S/D160G/A189R, K3E/H47V/P100E/D160G/E185A, K3E/H47W,K3E/H47W/T95S/P100E/L108V/A189R/T233S,K3E/H47W/P100E/L108V/S183L/E185A/A189R/M241A,K3E/H47W/P100E/E185A/A189R/I228L, K3E/H47W/L108V/T111K,K3E/H47W/S183L/A189R/I228L/T233S, K3E/H47W/A189R, K3E/H47W/I228L/T233S,K3E/T95S/P100E/D160G/I228L/T233S,K3E/T95S/P100E/S183L/V216M/I228L/T233S, K3E/T95S/P100E/S183L/T233S,K3E/T95S/E185A/A189R/V216M, K3E/T95S/A189R, K3E/T95S/T233S, K3E/D160G,K3E/S183L/E185A/A189R/I228L/T233S, K3E/S183L/A189R/I228L/T233S,K3E/E185A, K3E/E185A/A189R, K3E/A189R, V24L, V24L/K33Y/H47W,V24L/K33Y/H47W/I228L/M241A, V24L/K33Y/P100E/L108V/M241A,V24L/H47V/T95S/P100E, V24L/H47V/T95S/P100E/D160G/I228L/T233S/M241A,V24L/H47V/E185A/V216M/G218V, V24L/H47V/V216M, V24L/T95S/S183L,V24L/P100E/D160G/T233S, V24L/D160G/S183L/E185A, V24L/A189R/I228L/T233S,K33Y/H47V/T95S/P100E/T233S, K33Y/H47W/T95S/P100E/T233S/M241A,K33Y/H47W/D160G, K33Y/H47W/T233S, K33Y/P100E/S183L/E185A,K33Y/P100E/E185A/T233S, H47V, H47V/P100E/T111K/T233S, H47V/P100E/A189R,H47V/L108V/D160G/I228L/M241A, H47V/T111K, H47V/D160G/E185A/A189R/T233S,H47V/I228L/T233S, H47W/P100E/A189R/T233S, T95S/P100E/S183L,T95S/P100E/A189R, T95S/P100E/I228L, T95S/P100E/I228L/T233S,T95S/P100E/T233S, P100E/D160G/E185A, P100E/I228L/T233S, L108V,L108V/S183L/A189R/T233S, L108V/E185A/V216M/I228L/T233S, D160G/T233S,I228L, and I228L/T233S, wherein the amino acid positions of saidpolypeptide sequence are numbered with reference to SEQ ID NO: 246. Insome embodiments, the engineered uridine phosphorylase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 246. In some embodiments, the engineered uridine phosphorylasecomprises a polypeptide sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:246. In some embodiments, the engineered uridine phosphorylase comprisesa polypeptide sequence having at least 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 246.

In some embodiments, the polypeptide sequence has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 594, and wherein the polypeptide of theengineered uridine phosphorylase comprises at least one substitution orsubstitution set at one or more positions in said polypeptide sequenceselected from 6, 6/9/29/40/100/121/126/179/181/189/237, 6/9/121/179/181,6/46/52/63/97/121/126/179, 6/52/180/181, 6/63/126/179/242, 9,9/40/46/97/100/106/135/179/181/207/231, 9/52/126/189/242,9/97/100/106/126/180/207/231, 9/181/242, 29, 40, 46, 52, 52/63/126,52/179/189, 61, 63, 97, 100, 106, 121, 126, 126/180/189, 135, 142, 179,180, 181, 189, 201, 207, 230, 231, 236, 237, and 242, wherein the aminoacid positions of said polypeptide sequence are numbered with referenceto SEQ ID NO: 594. In some embodiments, the polypeptide sequence of theengineered uridine phosphorylase has at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 594, and wherein the polypeptide of theengineered uridine phosphorylase comprises at least one substitution orsubstitution set at one or more positions in said polypeptide sequenceselected from 6S, 6S/9M/29A/40N/100G/121G/126M/179R/181Q/189E/237V,6S/9M/121G/179R/181Q, 6S/46Q/52S/63V/97T/121G/126M/179R,6S/52S/180L/181Q, 6S/63V/126M/179R/242I, 9M,9M/40N/46Q/97T/100G/106E/135D/179R/181Q/207S/231E,9M/52S/126M/189E/242I, 9M/97T/100G/106E/126M/180L/207s/231E,9M/181Q/242I, 29A, 40N, 46Q, 52S, 52S/63V/126M, 52S/179R/189E, 61A, 63V,97T, 100G, 106E, 121G, 126M, 126M/180L/189E, 135D, 142A, 179R, 180L,181Q, 189D, 189E, 201L, 207S, 230D, 231E, 236E, 237V, and 242I, whereinthe amino acid positions of said polypeptide sequence are numbered withreference to SEQ ID NO: 594. In some embodiments, the polypeptidesequence of the engineered uridine phosphorylase has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 594, and wherein the polypeptide of theengineered uridine phosphorylase comprises at least one substitution orsubstitution set at one or more positions in said polypeptide sequenceselected from V6S,V6S/L9M/D29A/K40N/P100G/L121G/L126M/H179R/K181Q/R189E/T237V,V6S/L9M/L121G/H179R/K181Q, V6S/S46Q/T52S/I63V/A97T/L121G/L126M/H179R,V6S/T52S/F180L/K181Q, V6S/I63V/L126M/H179R/V242I, L9M,L9M/K40N/S46Q/A97T/P100G/D106E/E135D/H179R/K181Q/A207S/A231E,L9M/T52S/L126M/R189E/V242I,L9M/A97T/P100G/D106E/L126M/F180L/A207S/A231E, L9M/K181Q/V242I, D29A,K40N, S46Q, T52S, T52S/I63V/L126M, T52S/H179R/R189E, P61A, I63V, A97T,P100G, D106E, L121G, L126M, L126M/F180L/R189E, E135D, E142A, H179R,F180L, K181Q, R189D, R189E, T201L, A207S, N230D, A231E, Q236E, T237V,and V242I, wherein the amino acid positions of said polypeptide sequenceare numbered with reference to SEQ ID NO: 594. In some embodiments, theengineered uridine phosphorylase comprises a polypeptide sequence havingat least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more sequence identity to SEQ ID NO: 594. In someembodiments, the engineered uridine phosphorylase comprises apolypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more sequence identity to SEQ ID NO: 594. In someembodiments, the engineered uridine phosphorylase comprises apolypeptide sequence having at least 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 594.

In some embodiments, the polypeptide sequence has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 776, and wherein the polypeptide of theengineered uridine phosphorylase comprises at least one substitution orsubstitution set at one or more positions in said polypeptide sequenceselected from 3, 3/8/22/36/181/235/250, 6, 6/45/51/81/126/226/233,6/45/51/144, 6/45/51/188/189, 6/45/51/189/208, 6/45/51/189/228/236,6/45/51/208/233, 6/45/149/188/189/208/233/236, 6/51/126/144/208,6/51/126/189/208/233/236, 6/51/126/189/231/236, 6/51/126/189/233,6/51/188/189/208/226/228, 6/51/188/189/236, 6/51/189/208/231/233,6/51/208/226/233, 6/51/208/231, 6/126/188/231/233, 6/144/208,6/188/189/208/228/233, 8, 8/36/143/147/235, 8/36/181/235, 8/142/147,8/147/181/250, 8/147/235, 9, 19, 20, 22, 22/147/181/235/250, 24, 36,36/143/147/181, 36/143/147/235, 40, 41, 43, 45, 45/51,45/51/126/144/208/226/228, 45/51/144, 45/51/144/208/226/231/233,45/51/188/189, 45/51/189/233, 45/51/208/226/231, 45/51/208/233,45/51/226, 45/126/189, 45/126/189/208/226, 45/144/189/228,45/144/226/231/233, 45/188/189, 45/188/189/208/228, 45/188/189/226/228,45/188/189/231/233, 45/189, 45/189/208, 46, 51, 51/126/144/208,51/126/144/226/231/233/236, 51/126/208, 51/144, 51/144/226,51/188/189/228, 51/189, 51/189/208/226, 51/208, 51/233, 57, 58, 80,80/135/147, 81, 81/126/144/188/208/228, 86, 103, 126,126/144/188/189/226, 134, 135, 141, 142, 143, 143/147/235, 144,144/188/228, 146, 147, 149, 181, 188, 188/189/233, 189, 207, 208,208/226/233, 208/228, 208/231/233, 226, 228, 230, 231, 232, 233, 235,236, 240, and 250, wherein the amino acid positions of said polypeptidesequence are numbered with reference to SEQ ID NO: 776. In someembodiments, the polypeptide sequence of the engineered uridinephosphorylase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:776, and wherein the polypeptide of the engineered uridine phosphorylasecomprises at least one substitution or substitution set at one or morepositions in said polypeptide sequence selected from 3N,3N/8A/22G/36I/81R/235G/250A, 6T, 6T/45G/51I/81W/126Y/226G/233D,6T/45G/51I/144G, 6T/45G/51I/188R/189Y, 6T/45G/51I/189V/228M/236W,6T/45G/51I/189Y/208V, 6T/45G/51I/208V/233D,6T/45G/149T/188R/189Y/208V/233D/236G, 6T/51I/126Y/144G/208V,6T/51I/126Y/189V/233D, 6T/51I/126Y/189Y/208V/233D/236G,6T/51/126Y/189Y/231V/236W, 6T/51I/188R/189Y/208V/226G/228M,6T/51I/188R/189Y/236G, 6T/51I/189Y/208V/231V/233D,6T/51I/208V/226G/233D, 6T/51I/208V/231V, 6T/126Y/188R/231V/233D,6T/144G/208V, 6T/188R/189Y/208V/228M/233D, 8A, 8A/36I/143G/147M/235G,8A/36I/181R/235G, 8A/142L/147C, 8A/147C/181R/250A, 8A/147M/235G, 9V,19V, 20L, 22G, 22G/147C/181R/235G/250A, 24V, 36I, 36I/143G/147C/181R,36I/143G/147C/235G, 40L, 40V, 41G, 43P, 45G, 45G/51I,45G/51I/126Y/144G/208V/226G/228M, 45G/51I/144G,45G/51I/144G/208V/226G/231V/233D, 45G/51I/188R/189V, 45G/51I/189V/233D,45G/51I/208V/226G/231V, 45G/51I/208V/233D, 45G/51I/226G, 45G/126Y/189Y,45G/126Y/189Y/208V/226G, 45G/144G/189V/228M, 45G/144G/226G/231V/233D,45G/188R/189Y, 45G/188R/189Y/208V/228M, 45G/188R/189Y/226G/228M,45G/188R/189Y/231V/233D, 45G/189V, 45G/189V/208V, 45G/189Y, 45T, 46Q,51I, 51I/126Y/144G/208V, 51I/126Y/144G/226G/231V/233D/236W,51I/126Y/208V, 51I/144G, 51I/144G/226G, 51I/188R/189Y/228M,51I/189V/208V/226G, 51I/189Y, 51I/208V, 51I/233D, 57S, 57T, 58T, 80M,80M/135V/147C, 81W, 81W/126Y/144G/188R/208V/228M, 86L, 103G, 126L, 126Q,126V, 126Y, 126Y/144G/188R/189V/226G, 134L, 135V, 141L, 142I, 142L,143G, 143G/147C/235G, 144G, 144G/188R/228M, 146V, 147C, 147M, 149F,181R, 188R, 188R/189V/233D, 189T, 189V, 189Y, 207C, 207G, 208V,208V/226G/233D, 208V/228M, 208V/231V/233D, 226G, 228M, 230E, 231V, 232S,233D, 235G, 235P, 236A, 236G, 236I, 236W, 240F, 240W, and 250A, whereinthe amino acid positions of said polypeptide sequence are numbered withreference to SEQ ID NO: 776. In some embodiments, the polypeptidesequence of the engineered uridine phosphorylase has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 776, and wherein the polypeptide of theengineered uridine phosphorylase comprises at least one substitution orsubstitution set at one or more positions in said polypeptide sequenceselected from E3N, E3N/H8A/A22G/A36I/Q181R/K235G/R250A, S6T,S6T/A45G/T51I/L81W/M126Y/Q226G/S233D, S6T/A45G/T51I/A144G,S6T/A45G/T51I/Q188R/E189Y, S6T/A45G/T51I/E189V/L228M/Q236W,S6T/A45G/T51I/E189Y/S208V, S6T/A45G/T51I/S208V/S233D,S6T/A45G/A149T/Q188R/E189Y/S208V/S233D/Q236G,S6T/T51I/M126Y/A144G/S208V, S6T/T51I/M126Y/E189V/S233D,S6T/T51I/M126Y/E189Y/S208V/S233D/Q236G,S6T/T51I/M126Y/E189Y/A231V/Q236W,S6T/T51I/Q188R/E189Y/S208V/Q226G/L228M, S6T/T51I/Q188R/E189Y/Q236G,S6T/T51I/E189Y/S208V/A231V/S233D, S6T/T51I/S208V/Q226G/S233D,S6T/T51I/S208V/A231V, S6T/M126Y/Q188R/A231V/S233D, S6T/A144G/S208V,S6T/Q188R/E189Y/S208V/L228M/S233D, H8A, H8A/A36I/A143G/I147M/K235G,H8A/A36I/Q181R/K235G, H8A/E142L/I147C, H8A/I147C/Q181R/R250A,H8A/I147M/K235G, M9V, A19V, T20L, A22G, A22G/I147C/Q181R/K235G/R250A,L24V, A36I, A36I/A143G/I147C/Q181R, A36I/A143G/I147C/K235G, N40L, N40V,P41G, K43P, A45G, A45G/T51I, A45G/T51I/M126Y/A144G/S208V/Q226G/L228M,A45G/T51I/A144G, A45G/T51I/A144G/S208V/Q226G/A231V/S233D,A45G/T51I/Q188R/E189V, A45G/T51I/E189V/S233D,A45G/T51I/S208V/Q226G/A231V, A45G/T51I/S208V/S233D, A45G/T51I/Q226G,A45G/M126Y/E189Y, A45G/M126Y/E189Y/S208V/Q226G, A45G/A144G/E189V/L228M,A45G/A144G/Q226G/A231V/S233D, A45G/Q188R/E189Y,A45G/Q188R/E189Y/S208V/L228M, A45G/Q188R/E189Y/Q226G/L228M,A45G/Q188R/E189Y/A231V/S233D, A45G/E189V, A45G/E189V/S208V, A45G/E189Y,A45T, S46Q, T51I, T51I/M126Y/A144G/S208V,T51I/M126Y/A144G/Q226G/A231V/S233D/Q236W, T51I/M126Y/S208V, T51I/A144G,T51I/A144G/Q226G, T51I/Q188R/E189Y/L228M, T51I/E189V/S208V/Q226G,T51I/E189Y, T51I/S208V, T51I/S233D, L57S, L57T, D58T, I80M,I80M/E135V/I147C, L81W, L81W/M126Y/A144G/Q188R/S208V/L228M, I86L, N103G,M126L, M126Q, M126V, M126Y, M126Y/A144G/Q188R/E189V/Q226G, F134L, E135V,V141L, E142I, E142L, A143G, A143G/I147C/K235G, A144G, A144G/Q188R/L228M,S146V, I147C, I147M, A149F, Q181R, Q188R, Q188R/E189V/S233D, E189T,E189V, E189Y, A207C, A207G, S208V, S208V/Q226G/S233D, S208V/L228M,S208V/A231V/S233D, Q226G, L228M, N230E, A231V, E232S, S233D, K235G,K235P, Q236A, Q236G, Q236I, Q236W, H240F, H240W, and R250A, wherein theamino acid positions of said polypeptide sequence are numbered withreference to SEQ ID NO: 776. In some embodiments, the engineered uridinephosphorylase comprises a polypeptide sequence having at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 776. In some embodiments, the engineereduridine phosphorylase comprises a polypeptide sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 776. In some embodiments, the engineered uridinephosphorylase comprises a polypeptide sequence having at least 95%, 96%,97%, 98%, 99%, or more sequence identity to SEQ ID NO: 776.

In some embodiments, the polypeptide sequence has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 868, and wherein the polypeptide of theengineered uridine phosphorylase comprises at least one substitution orsubstitution set at one or more positions in said polypeptide sequenceselected from 6, 6/8/9/126/147, 6/9/24/181/189, 6/9/181/189/235,6/9/208/233/235, 6/24, 6/24/43/46/181/189, 6/24/43/126/147/189,6/24/46/103/181/208, 6/24/46/147/240, 6/24/103/189, 6/24/126/189,6/24/147, 6/46/126/147/181/235/240, 6/46/147/189/240, 6/103,6/103/147/230/233, 6/103/189/235, 6/126/181/189, 6/126/181/189/235,6/126/233/235, 6/181/230/233/235, 9/43/46/103/189/233/240,9/46/126/147/181, 9/46/147/233, 24/43/46/147/230/235, 24/46/126,24/46/147/189, 24/46/208/230/233/235, 24/103/126/147,24/103/126/147/181/189/208/233, 24/147, 24/147/189/230/233,24/181/189/230/233/235, 24/189/230, 24/208, 43/46/126/147/189/240,43/103/189/208/233, 103/126/189/233/235, 103/147/181, 147/181/233, 189,189/235, and 208/233, wherein the amino acid positions of saidpolypeptide sequence are numbered with reference to SEQ ID NO: 868. Insome embodiments, the polypeptide sequence of the engineered uridinephosphorylase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:868, and wherein the polypeptide of the engineered uridine phosphorylasecomprises at least one substitution or substitution set at one or morepositions in said polypeptide sequence selected 6T, 6T/8A/9V/126Y/147C,6T/9V/24V/181R/189E, 6T/9V/181R/189E/235P, 6T/9V/208V/233D/235P, 6T/24V,6T/24V/43P/46Q/181R/189E, 6T/24V/43P/126Y/147C/189E,6T/24V/46Q/103G/181R/208V, 6T/24V/46Q/147C/240F, 6T/24V/103G/189E,6T/24V/126Y/189E, 6T/24V/147C, 6T/46Q/126Y/147C/181R/235P/240F,6T/46Q/147C/189E/240F, 6T/103G, 6T/103G/147C/230E/233D,6T/103G/189E/235P, 6T/126Y/181R/189E, 6T/126Y/181R/189E/235P,6T/126Y/233D/235P, 6T/181R/230E/233D/235P,9V/43P/46Q/103G/189E/233D/240W, 9V/46Q/126Y/147C/181R, 9V/46Q/147C/233D,24V/43P/46Q/147C/230E/235P, 24V/46Q/126Y, 24V/46Q/147C/189E,24V/46Q/208V/230E/233D/235P, 24V/103G/126Y/147C,24V/103G/126Y/147C/181R/189E/208V/233D, 24V/147C,24V/147C/189E/230E/233D, 24V/181R/189E/230E/233D/235P, 24V/189E/230E,24V/208V, 43P/46Q/126Y/147C/189E/240F, 43P/103G/189E/208V/233D,103G/126Y/189E/233D/235P, 103G/147C/181R, 147C/181R/233D, 189E,189E/235P, and 208V/233D, wherein the amino acid positions of saidpolypeptide sequence are numbered with reference to SEQ ID NO: 868. Insome embodiments, the polypeptide sequence of the engineered uridinephosphorylase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:868, and wherein the polypeptide of the engineered uridine phosphorylasecomprises at least one substitution or substitution set at one or morepositions in said polypeptide sequence selected from S6T,S6T/H8A/M9V/M126Y/I147C, S6T/M9V/L24V/Q181R/V189E,S6T/M9V/Q181R/V189E/K235P, S6T/M9V/S208V/S233D/K235P, S6T/L24V,S6T/L24V/K43P/S46Q/Q181R/V189E, S6T/L24V/K43P/M126Y/I147C/V189E,S6T/L24V/S46Q/N103G/Q181R/S208V, S6T/L24V/S46Q/I147C/H240F,S6T/L24V/N103G/V189E, S6T/L24V/M126Y/V189E, S6T/L24V/I147C,S6T/S46Q/M126Y/I147C/Q181R/K235P/H240F, S6T/S46Q/I147C/V189E/H240F,S6T/N103G, S6T/N103G/I147C/N230E/S233D, S6T/N103G/V189E/K235P,S6T/M126Y/Q181R/V189E, S6T/M126Y/Q181R/V189E/K235P,S6T/M126Y/S233D/K235P, S6T/Q181R/N230E/S233D/K235P,M9V/K43P/S46Q/N103G/V189E/S233D/H240W, M9V/S46Q/M126Y/I147C/Q181R,M9V/S46Q/I147C/S233D, L24V/K43P/S46Q/I147C/N230E/K235P, L24V/S46Q/M126Y,L24V/S46Q/I147C/V189E, L24V/S46Q/S208V/N230E/S233D/K235P,L24V/N103G/M126Y/I147C, L24V/N103G/M126Y/I147C/Q181R/V189E/S208V/S233D,L24V/I147C, L24V/I147C/V189E/N230E/S233D,L24V/Q181R/V189E/N230E/S233D/K235P, L24V/V189E/N230E, L24V/S208V,K43P/S46Q/M126Y/I147C/V189E/H240F, K43P/N103G/V189E/S208V/S233D,N103G/M126Y/V189E/S233D/K235P, N103G/I147C/Q181R, I147C/Q181R/S233D,V189E, V189E/K235P, and S208V/S233D, wherein the amino acid positions ofsaid polypeptide sequence are numbered with reference to SEQ ID NO: 868.In some embodiments, the engineered uridine phosphorylase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 868. In some embodiments, the engineered uridine phosphorylasecomprises a polypeptide sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:868. In some embodiments, the engineered uridine phosphorylase comprisesa polypeptide sequence having at least 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 868.

In some additional embodiments, the present invention providesengineered uridine phosphorylases, wherein the engineered uridinephosphorylases comprise polypeptide sequences that are at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the sequence of at least one engineered uridinephosphorylase variant set forth in Table 1.2, 2.2, 3.1, 4.1, 5.2 and/or6.1.

In some additional embodiments, the present invention providesengineered uridine phosphorylases, wherein the engineered uridinephosphorylases comprise polypeptide sequences that are at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to SEQ ID NOs: 2, 4, 246, 594, 776, and/or 868. In someembodiments, the engineered uridine phosphorylase comprises a variantengineered uridine phosphorylase set forth in SEQ ID NOs: 4, 246, 594,776, and/or 868.

The present invention also provides engineered uridine phosphorylaseswherein the engineered uridine phosphorylases comprise polypeptidesequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of atleast one engineered uridine phosphorylase variant set forth in the evennumbered sequences of SEQ ID NOs: 4-1196.

The present invention further provides engineered uridinephosphorylases, wherein said engineered uridine phosphorylases compriseat least one improved property compared to wild-type Escherichia coliuridine phosphorylase. In some embodiments, the improved propertycomprises improved activity on a substrate. In some further embodiments,the substrates comprise 5′-isobutyryl ribose-1-phosphate (compound (2))and uracil (compound (3)). In some additional embodiments, the improvedproperty comprises improved production of compound (1) from compound (2)and compound (3). In yet some additional embodiments, the engineereduridine phosphorylase is purified. The present invention also providescompositions comprising at least one engineered uridine phosphorylaseprovided herein.

The present invention also provides polynucleotide sequences encoding atleast one engineered uridine phosphorylase provided herein. In someembodiments, the polynucleotide sequence encoding at least oneengineered uridine phosphorylase comprises a polynucleotide sequencehaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NOs: 1, 3, 245,593, 775 and/or 867. In some embodiments, the polynucleotide sequenceencoding at least one engineered uridine phosphorylase, comprises apolynucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NOs: 1, 3, 245, 593, 775 and/or 867, wherein thepolynucleotide sequence of said engineered uridine phosphorylasecomprises at least one substitution at one or more positions. In somefurther embodiments, the polynucleotide sequence encoding at least oneengineered uridine phosphorylase comprises at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NOs: 1, 3, 245, 593, 775 and/or 867, or a functionalfragment thereof. In yet some additional embodiments, the polynucleotidesequence is operably linked to a control sequence. In some furtherembodiments, the polynucleotide sequence is codon optimized. In stillsome additional embodiments, the polynucleotide sequence comprises apolynucleotide sequence forth in the odd numbered sequences of SEQ IDNOs: 1, 3, 245, 593, 775 and/or 867.

The present invention also provides expression vectors comprising atleast one polynucleotide sequence provided herein. The present inventionfurther provides host cells comprising at least one expression vectorprovided herein. In some embodiments, the present invention provideshost cells comprising at least one polynucleotide sequence providedherein.

The present invention also provides methods of producing an engineereduridine phosphorylase in a host cell, comprising culturing the host cellprovided herein, under suitable conditions, such that at least oneengineered uridine phosphorylase is produced. In some embodiments, themethods further comprise recovering at least one engineered uridinephosphorylase from the culture and/or host cell. In some additionalembodiments, the methods further comprise the step of purifying said atleast one engineered uridine phosphorylase.

DESCRIPTION OF THE INVENTION

The present invention provides engineered uridine phosphorylase (UP)enzymes, polypeptides having UP activity, and polynucleotides encodingthese enzymes, as well as vectors and host cells comprising thesepolynucleotides and polypeptides. Methods for producing UP enzymes arealso provided. The present invention further provides compositionscomprising the UP enzymes and methods of using the engineered UPenzymes. The present invention finds particular use in the production ofpharmaceutical compounds.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention pertains. Generally,the nomenclature used herein and the laboratory procedures of cellculture, molecular genetics, microbiology, organic chemistry, analyticalchemistry and nucleic acid chemistry described below are thosewell-known and commonly employed in the art. Such techniques arewell-known and described in numerous texts and reference works wellknown to those of skill in the art. Standard techniques, ormodifications thereof, are used for chemical syntheses and chemicalanalyses. All patents, patent applications, articles and publicationsmentioned herein, both supra and infra, are hereby expresslyincorporated herein by reference.

Although any suitable methods and materials similar or equivalent tothose described herein find use in the practice of the presentinvention, some methods and materials are described herein. It is to beunderstood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary,depending upon the context they are used by those of skill in the art.Accordingly, the terms defined immediately below are more fullydescribed by reference to the invention as a whole.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the present invention. The section headingsused herein are for organizational purposes only and not to be construedas limiting the subject matter described. Numeric ranges are inclusiveof the numbers defining the range. Thus, every numerical range disclosedherein is intended to encompass every narrower numerical range thatfalls within such broader numerical range, as if such narrower numericalranges were all expressly written herein. It is also intended that everymaximum (or minimum) numerical limitation disclosed herein includesevery lower (or higher) numerical limitation, as if such lower (orhigher) numerical limitations were expressly written herein.

Abbreviations and Definitions

The abbreviations used for the genetically encoded amino acids areconventional and are as follows: alanine (Ala or A), arginine (Arg orR), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C),glutamate (Glu or E), glutamine (Gln or Q), histidine (His or H),isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine(Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser orS), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y),and valine (Val or V).

When the three-letter abbreviations are used, unless specificallypreceded by an “L” or a “D” or clear from the context in which theabbreviation is used, the amino acid may be in either the L- orD-configuration about α-carbon (Cα). For example, whereas “Ala”designates alanine without specifying the configuration about theα-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine,respectively. When the one-letter abbreviations are used, upper caseletters designate amino acids in the L-configuration about the α-carbonand lower case letters designate amino acids in the D-configurationabout the α-carbon. For example, “A” designates L-alanine and “a”designates D-alanine. When polypeptide sequences are presented as astring of one-letter or three-letter abbreviations (or mixturesthereof), the sequences are presented in the amino (N) to carboxy (C)direction in accordance with common convention.

The abbreviations used for the genetically encoding nucleosides areconventional and are as follows: adenosine (A); guanosine (G); cytidine(C); thymidine (T); and uridine (U). Unless specifically delineated, theabbreviated nucleosides may be either ribonucleosides or2′-deoxyribonucleosides. The nucleosides may be specified as beingeither ribonucleosides or 2′-deoxyribonucleosides on an individual basisor on an aggregate basis. When nucleic acid sequences are presented as astring of one-letter abbreviations, the sequences are presented in the5′ to 3′ direction in accordance with common convention, and thephosphates are not indicated.

In reference to the present invention, the technical and scientificterms used in the descriptions herein will have the meanings commonlyunderstood by one of ordinary skill in the art, unless specificallydefined otherwise. Accordingly, the following terms are intended to havethe following meanings.

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “a polypeptide” includes more than onepolypeptide.

Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,”and “including” are interchangeable and not intended to be limiting.Thus, as used herein, the term “comprising” and its cognates are used intheir inclusive sense (i.e., equivalent to the term “including” and itscorresponding cognates).

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

As used herein, the term “about” means an acceptable error for aparticular value. In some instances, “about” means within 0.05%, 0.5%,1.0%, or 2.0%, of a given value range. In some instances, “about” meanswithin 1, 2, 3, or 4 standard deviations of a given value.

As used herein, “EC” number refers to the Enzyme Nomenclature of theNomenclature Committee of the International Union of Biochemistry andMolecular Biology (NC-IUBMB). The IUBMB biochemical classification is anumerical classification system for enzymes based on the chemicalreactions they catalyze.

As used herein, “ATCC” refers to the American Type Culture Collectionwhose biorepository collection includes genes and strains.

As used herein, “NCBI” refers to National Center for BiologicalInformation and the sequence databases provided therein.

As used herein, “uridine phosphorylase” (“UP”) enzymes are enzymes thatcatalyze the reversible conversion of 5′-isobutyryl ribose-1-phosphate(compound (2)) and uracil (compound (3)) and/or related compounds to5′-isobutyryluridine (compound (1)) and/or related compounds. UP enzymesmay be naturally occurring, including the wild-type UP enzyme ofEscherichia coli or other uridine phosphorylases ornucleosidetransferases found in humans, bacteria, fungi, plants or otherspecies, or the UP enzymes may be engineered polypeptides generated byhuman manipulation.

“Protein,” “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation or phosphorylation). Included within thisdefinition are D- and L-amino acids, and mixtures of D- and L-aminoacids, as well as polymers comprising D- and L-amino acids, and mixturesof D- and L-amino acids.

“Amino acids” are referred to herein by either their commonly knownthree-letter symbols or by the one-letter symbols recommended byIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single letter codes.

As used herein, “hydrophilic amino acid or residue” refers to an aminoacid or residue having a side chain exhibiting a hydrophobicity of lessthan zero according to the normalized consensus hydrophobicity scale ofEisenberg et al., (Eisenberg et al., J. Mol. Biol., 179:125-142 [1984]).Genetically encoded hydrophilic amino acids include L-Thr (T), L-Ser(S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q), L-Asp (D), L-Lys (K)and L-Arg (R).

As used herein, “acidic amino acid or residue” refers to a hydrophilicamino acid or residue having a side chain exhibiting a pKa value of lessthan about 6 when the amino acid is included in a peptide orpolypeptide. Acidic amino acids typically have negatively charged sidechains at physiological pH due to loss of a hydrogen ion. Geneticallyencoded acidic amino acids include L-Glu (E) and L-Asp (D).

As used herein, “basic amino acid or residue” refers to a hydrophilicamino acid or residue having a side chain exhibiting a pKa value ofgreater than about 6 when the amino acid is included in a peptide orpolypeptide. Basic amino acids typically have positively charged sidechains at physiological pH due to association with hydronium ion.Genetically encoded basic amino acids include L-Arg (R) and L-Lys (K).

As used herein, “polar amino acid or residue” refers to a hydrophilicamino acid or residue having a side chain that is uncharged atphysiological pH, but which has at least one bond in which the pair ofelectrons shared in common by two atoms is held more closely by one ofthe atoms. Genetically encoded polar amino acids include L-Asn (N),L-Gln (Q), L-Ser (S) and L-Thr (T).

As used herein, “hydrophobic amino acid or residue” refers to an aminoacid or residue having a side chain exhibiting a hydrophobicity ofgreater than zero according to the normalized consensus hydrophobicityscale of Eisenberg et al., (Eisenberg et al., J. Mol. Biol., 179:125-142[1984]). Genetically encoded hydrophobic amino acids include L-Pro (P),L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala(A) and L-Tyr (Y).

As used herein, “aromatic amino acid or residue” refers to a hydrophilicor hydrophobic amino acid or residue having a side chain that includesat least one aromatic or heteroaromatic ring. Genetically encodedaromatic amino acids include L-Phe (F), L-Tyr (Y) and L-Trp (W).Although owing to the pKa of its heteroaromatic nitrogen atom L-His (H)it is sometimes classified as a basic residue, or as an aromatic residueas its side chain includes a heteroaromatic ring, herein histidine isclassified as a hydrophilic residue or as a “constrained residue” (seebelow).

As used herein, “constrained amino acid or residue” refers to an aminoacid or residue that has a constrained geometry. Herein, constrainedresidues include L-Pro (P) and L-His (H). Histidine has a constrainedgeometry because it has a relatively small imidazole ring. Proline has aconstrained geometry because it also has a five membered ring.

As used herein, “non-polar amino acid or residue” refers to ahydrophobic amino acid or residue having a side chain that is unchargedat physiological pH and which has bonds in which the pair of electronsshared in common by two atoms is generally held equally by each of thetwo atoms (i.e., the side chain is not polar). Genetically encodednon-polar amino acids include L-Gly (G), L-Leu (L), L-Val (V), L-Ile(I), L-Met (M) and L-Ala (A).

As used herein, “aliphatic amino acid or residue” refers to ahydrophobic amino acid or residue having an aliphatic hydrocarbon sidechain. Genetically encoded aliphatic amino acids include L-Ala (A),L-Val (V), L-Leu (L) and L-Ile (I). It is noted that cysteine (or“L-Cys” or [Cl]) is unusual in that it can form disulfide bridges withother L-Cys (C) amino acids or other sulfanyl- or sulfhydryl-containingamino acids. The “cysteine-like residues” include cysteine and otheramino acids that contain sulfhydryl moieties that are available forformation of disulfide bridges. The ability of L-Cys (C) (and otheramino acids with -SH containing side chains) to exist in a peptide ineither the reduced free —SH or oxidized disulfide-bridged form affectswhether L-Cys (C) contributes net hydrophobic or hydrophilic characterto a peptide. While L-Cys (C) exhibits a hydrophobicity of 0.29according to the normalized consensus scale of Eisenberg (Eisenberg etal., 1984, supra), it is to be understood that for purposes of thepresent disclosure, L-Cys (C) is categorized into its own unique group.

As used herein, “small amino acid or residue” refers to an amino acid orresidue having a side chain that is composed of a total three or fewercarbon and/or heteroatoms (excluding the a-carbon and hydrogens). Thesmall amino acids or residues may be further categorized as aliphatic,non-polar, polar or acidic small amino acids or residues, in accordancewith the above definitions. Genetically-encoded small amino acidsinclude L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T)and L-Asp (D).

As used herein, “hydroxyl-containing amino acid or residue” refers to anamino acid containing a hydroxyl (-OH) moiety. Genetically-encodedhydroxyl-containing amino acids include L-Ser (S) L-Thr (T) and L-Tyr(Y).

As used herein, “polynucleotide” and “nucleic acid” refer to two or morenucleotides that are covalently linked together. The polynucleotide maybe wholly comprised of ribonucleotides (i.e., RNA), wholly comprised of2′ deoxyribonucleotides (i.e., DNA), or comprised of mixtures of ribo-and 2′ deoxyribonucleotides. While the nucleosides will typically belinked together via standard phosphodiester linkages, thepolynucleotides may include one or more non-standard linkages Thepolynucleotide may be single-stranded or double-stranded, or may includeboth single-stranded regions and double-stranded regions. Moreover,while a polynucleotide will typically be composed of the naturallyoccurring encoding nucleobases (i.e., adenine, guanine, uracil, thymineand cytosine), it may include one or more modified and/or syntheticnucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc.In some embodiments, such modified or synthetic nucleobases arenucleobases encoding amino acid sequences.

As used herein, “nucleoside” refers to glycosylamines comprising anucleobase (i.e., a nitrogenous base), and a 5-carbon sugar (e.g.,ribose or deoxyribose). Non-limiting examples of nucleosides includecytidine, uridine, adenosine, guanosine, thymidine, and inosine. Incontrast, the term “nucleotide” refers to the glycosylamines comprisinga nucleobase, a 5-carbon sugar, and one or more phosphate groups. Insome embodiments, nucleosides can be phosphorylated by kinases toproduce nucleotides.

As used herein, “nucleoside diphosphate” refers to glycosylaminescomprising a nucleobase (i.e., a nitrogenous base), a 5-carbon sugar(e.g., ribose or deoxyribose), and a diphosphate (i.e., pyrophosphate)moiety. In some embodiments herein, “nucleoside diphosphate” isabbreviated as “NDP”. Non-limiting examples of nucleoside diphosphatesinclude cytidine diphosphate (CDP), uridine diphosphate (UDP), adenosinediphosphate (ADP), guanosine diphosphate (GDP), thymidine diphosphate(TDP), and inosine diphosphate (IDP). The terms “nucleoside” and“nucleotide” may be used interchangeably in some contexts.

As used herein, “coding sequence” refers to that portion of a nucleicacid (e.g., a gene) that encodes an amino acid sequence of a protein.

As used herein, the terms “biocatalysis,” “biocatalytic,”“biotransformation,” and “biosynthesis” refer to the use of enzymes toperform chemical reactions on organic compounds.

As used herein, “wild-type” and “naturally occurring” refer to the formfound in nature. For example, a wild-type polypeptide or polynucleotidesequence is a sequence present in an organism that can be isolated froma source in nature and which has not been intentionally modified byhuman manipulation.

As used herein, “recombinant,” “engineered,” “variant,” and“non-naturally occurring” when used with reference to a cell, nucleicacid, or polypeptide, refers to a material, or a material correspondingto the natural or native form of the material, that has been modified ina manner that would not otherwise exist in nature. In some embodiments,the cell, nucleic acid or polypeptide is identical a naturally occurringcell, nucleic acid or polypeptide, but is produced or derived fromsynthetic materials and/or by manipulation using recombinant techniques.Non-limiting examples include, among others, recombinant cellsexpressing genes that are not found within the native (non-recombinant)form of the cell or express native genes that are otherwise expressed ata different level.

The term “percent (%) sequence identity” is used herein to refer tocomparisons among polynucleotides or polypeptides, and are determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence for optimal alignment of the twosequences. The percentage may be calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. Alternatively, thepercentage may be calculated by determining the number of positions atwhich either the identical nucleic acid base or amino acid residueoccurs in both sequences or a nucleic acid base or amino acid residue isaligned with a gap to yield the number of matched positions, dividingthe number of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Those of skill in the art appreciatethat there are many established algorithms available to align twosequences. Optimal alignment of sequences for comparison can beconducted by any suitable method, including, but not limited to thelocal homology algorithm of Smith and Waterman (Smith and Waterman, Adv.Appl. Math., 2:482 [1981]), by the homology alignment algorithm ofNeedleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443[1970]), by the search for similarity method of Pearson and Lipman(Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]), bycomputerized implementations of these algorithms (e.g., GAP, BESTFIT,FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visualinspection, as known in the art. Examples of algorithms that aresuitable for determining percent sequence identity and sequencesimilarity include, but are not limited to the BLAST and BLAST 2.0algorithms, which are described by Altschul et al. (See Altschul et al.,J. Mol. Biol., 215: 403-410 [1990]; and Altschul et al., Nucl. AcidsRes., 3389-3402 [1977], respectively). Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information website. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as, theneighborhood word score threshold (See, Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always>0) and N (penalty score formismatching residues; always<0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word length(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix(See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915[1989]). Exemplary determination of sequence alignment and % sequenceidentity can employ the BESTFIT or GAP programs in the GCG WisconsinSoftware package (Accelrys, Madison Wis.), using default parametersprovided.

As used herein, “reference sequence” refers to a defined sequence usedas a basis for a sequence and/or activity comparison. A referencesequence may be a subset of a larger sequence, for example, a segment ofa full-length gene or polypeptide sequence. Generally, a referencesequence is at least 20 nucleotide or amino acid residues in length, atleast 25 residues in length, at least 50 residues in length, at least100 residues in length or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptides aretypically performed by comparing sequences of the two polynucleotides orpolypeptides over a “comparison window” to identify and compare localregions of sequence similarity. In some embodiments, a “referencesequence” can be based on a primary amino acid sequence, where thereference sequence is a sequence that can have one or more changes inthe primary sequence.

As used herein, “comparison window” refers to a conceptual segment of atleast about 20 contiguous nucleotide positions or amino acid residueswherein a sequence may be compared to a reference sequence of at least20 contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

As used herein, “corresponding to,” “reference to,” and “relative to”when used in the context of the numbering of a given amino acid orpolynucleotide sequence refer to the numbering of the residues of aspecified reference sequence when the given amino acid or polynucleotidesequence is compared to the reference sequence. In other words, theresidue number or residue position of a given polymer is designated withrespect to the reference sequence rather than by the actual numericalposition of the residue within the given amino acid or polynucleotidesequence. For example, a given amino acid sequence, such as that of anengineered uridine phosphorylase, can be aligned to a reference sequenceby introducing gaps to optimize residue matches between the twosequences. In these cases, although the gaps are present, the numberingof the residue in the given amino acid or polynucleotide sequence ismade with respect to the reference sequence to which it has beenaligned.

As used herein, “substantial identity” refers to a polynucleotide orpolypeptide sequence that has at least 80 percent sequence identity, atleast 85 percent identity, at least between 89 to 95 percent sequenceidentity, or more usually, at least 99 percent sequence identity ascompared to a reference sequence over a comparison window of at least 20residue positions, frequently over a window of at least 30-50 residues,wherein the percentage of sequence identity is calculated by comparingthe reference sequence to a sequence that includes deletions oradditions which total 20 percent or less of the reference sequence overthe window of comparison. In some specific embodiments applied topolypeptides, the term “substantial identity” means that two polypeptidesequences, when optimally aligned, such as by the programs GAP orBESTFIT using default gap weights, share at least 80 percent sequenceidentity, preferably at least 89 percent sequence identity, at least 95percent sequence identity or more (e.g., 99 percent sequence identity).In some embodiments, residue positions that are not identical insequences being compared differ by conservative amino acidsubstitutions.

As used herein, “amino acid difference” and “residue difference” referto a difference in the amino acid residue at a position of a polypeptidesequence relative to the amino acid residue at a corresponding positionin a reference sequence. In some cases, the reference sequence has ahistidine tag, but the numbering is maintained relative to theequivalent reference sequence without the histidine tag. The positionsof amino acid differences generally are referred to herein as “Xn,”where n refers to the corresponding position in the reference sequenceupon which the residue difference is based. For example, a “residuedifference at position X93 as compared to SEQ ID NO:4” refers to adifference of the amino acid residue at the polypeptide positioncorresponding to position 93 of SEQ ID NO:4. Thus, if the referencepolypeptide of SEQ ID NO:4 has a serine at position 93, then a “residuedifference at position X93 as compared to SEQ ID NO:4” an amino acidsubstitution of any residue other than serine at the position of thepolypeptide corresponding to position 93 of SEQ ID NO:4. In mostinstances herein, the specific amino acid residue difference at aposition is indicated as “XnY” where “Xn” specified the correspondingposition as described above, and “Y” is the single letter identifier ofthe amino acid found in the engineered polypeptide (i.e., the differentresidue than in the reference polypeptide). In some instances (e.g., inthe Tables presented in the Examples), the present invention alsoprovides specific amino acid differences denoted by the conventionalnotation “AnB”, where A is the single letter identifier of the residuein the reference sequence, “n” is the number of the residue position inthe reference sequence, and B is the single letter identifier of theresidue substitution in the sequence of the engineered polypeptide. Insome instances, a polypeptide of the present invention can include oneor more amino acid residue differences relative to a reference sequence,which is indicated by a list of the specified positions where residuedifferences are present relative to the reference sequence. In someembodiments, where more than one amino acid can be used in a specificresidue position of a polypeptide, the various amino acid residues thatcan be used are separated by a “/” (e.g., X307H/X307P or X307H/P). Theslash may also be used to indicate multiple substitutions within a givenvariant (i.e., there is more than one substitution present in a givensequence, such as in a combinatorial variant). In some embodiments, thepresent invention includes engineered polypeptide sequences comprisingone or more amino acid differences comprising conservative ornon-conservative amino acid substitutions. In some additionalembodiments, the present invention provides engineered polypeptidesequences comprising both conservative and non-conservative amino acidsubstitutions.

As used herein, “conservative amino acid substitution” refers to asubstitution of a residue with a different residue having a similar sidechain, and thus typically involves substitution of the amino acid in thepolypeptide with amino acids within the same or similar defined class ofamino acids. By way of example and not limitation, in some embodiments,an amino acid with an aliphatic side chain is substituted with anotheraliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine);an amino acid with an hydroxyl side chain is substituted with anotheramino acid with an hydroxyl side chain (e.g., serine and threonine); anamino acids having aromatic side chains is substituted with anotheramino acid having an aromatic side chain (e.g., phenylalanine, tyrosine,tryptophan, and histidine); an amino acid with a basic side chain issubstituted with another amino acid with a basis side chain (e.g.,lysine and arginine); an amino acid with an acidic side chain issubstituted with another amino acid with an acidic side chain (e.g.,aspartic acid or glutamic acid); and/or a hydrophobic or hydrophilicamino acid is replaced with another hydrophobic or hydrophilic aminoacid, respectively.

As used herein, “non-conservative substitution” refers to substitutionof an amino acid in the polypeptide with an amino acid withsignificantly differing side chain properties. Non-conservativesubstitutions may use amino acids between, rather than within, thedefined groups and affects (a) the structure of the peptide backbone inthe area of the substitution (e.g., proline for glycine) (b) the chargeor hydrophobicity, or (c) the bulk of the side chain. By way of exampleand not limitation, an exemplary non-conservative substitution can be anacidic amino acid substituted with a basic or aliphatic amino acid; anaromatic amino acid substituted with a small amino acid; and ahydrophilic amino acid substituted with a hydrophobic amino acid.

As used herein, “deletion” refers to modification to the polypeptide byremoval of one or more amino acids from the reference polypeptide.Deletions can comprise removal of 1 or more amino acids, 2 or more aminoacids, 5 or more amino acids, 10 or more amino acids, 15 or more aminoacids, or 20 or more amino acids, up to 10% of the total number of aminoacids, or up to 20% of the total number of amino acids making up thereference enzyme while retaining enzymatic activity and/or retaining theimproved properties of an engineered uridine phosphorylase enzyme.Deletions can be directed to the internal portions and/or terminalportions of the polypeptide. In various embodiments, the deletion cancomprise a continuous segment or can be discontinuous. Deletions aretypically indicated by “-” in amino acid sequences.

As used herein, “insertion” refers to modification to the polypeptide byaddition of one or more amino acids from the reference polypeptide.Insertions can be in the internal portions of the polypeptide, or to thecarboxy or amino terminus. Insertions as used herein include fusionproteins as is known in the art. The insertion can be a contiguoussegment of amino acids or separated by one or more of the amino acids inthe naturally occurring polypeptide.

The term “amino acid substitution set” or “substitution set” refers to agroup of amino acid substitutions in a polypeptide sequence, as comparedto a reference sequence. A substitution set can have 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. Insome embodiments, a substitution set refers to the set of amino acidsubstitutions that is present in any of the variant uridinephosphorylases listed in the Tables provided in the Examples

A “functional fragment” and “biologically active fragment” are usedinterchangeably herein to refer to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion(s) and/or internaldeletions, but where the remaining amino acid sequence is identical tothe corresponding positions in the sequence to which it is beingcompared (e.g., a full-length engineered uridine phosphorylase of thepresent invention) and that retains substantially all of the activity ofthe full-length polypeptide.

As used herein, “isolated polypeptide” refers to a polypeptide which issubstantially separated from other contaminants that naturally accompanyit (e.g., protein, lipids, and polynucleotides). The term embracespolypeptides which have been removed or purified from their naturallyoccurring environment or expression system (e.g., within a host cell orvia in vitro synthesis). The recombinant uridine phosphorylasepolypeptides may be present within a cell, present in the cellularmedium, or prepared in various forms, such as lysates or isolatedpreparations. As such, in some embodiments, the recombinant uridinephosphorylase polypeptides can be an isolated polypeptide.

As used herein, “substantially pure polypeptide” or “purified protein”refers to a composition in which the polypeptide species is thepredominant species present (i.e., on a molar or weight basis it is moreabundant than any other individual macromolecular species in thecomposition), and is generally a substantially purified composition whenthe object species comprises at least about 50 percent of themacromolecular species present by mole or % weight. However, in someembodiments, the composition comprising uridine phosphorylase comprisesuridine phosphorylase that is less than 50% pure (e.g., about 10%, about20%, about 30%, about 40%, or about 50%) Generally, a substantially pureuridine phosphorylase composition comprises about 60% or more, about 70%or more, about 80% or more, about 90% or more, about 95% or more, andabout 98% or more of all macromolecular species by mole or % weightpresent in the composition. In some embodiments, the object species ispurified to essential homogeneity (i.e., contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.Solvent species, small molecules (<500 Daltons), and elemental ionspecies are not considered macromolecular species. In some embodiments,the isolated recombinant uridine phosphorylase polypeptides aresubstantially pure polypeptide compositions.

As used herein, “improved enzyme property” refers to at least oneimproved property of an enzyme. In some embodiments, the presentinvention provides engineered uridine phosphorylase polypeptides thatexhibit an improvement in any enzyme property as compared to a referenceuridine phosphorylase polypeptide and/or a wild-type uridinephosphorylase polypeptide, and/or another engineered uridinephosphorylase polypeptide. Thus, the level of “improvement” can bedetermined and compared between various uridine phosphorylasepolypeptides, including wild-type, as well as engineered uridinephosphorylases. Improved properties include, but are not limited, tosuch properties as increased protein expression, increasedthermoactivity, increased thermostability, increased pH activity,increased stability, increased enzymatic activity, increased substratespecificity or affinity, increased specific activity, increasedresistance to substrate or end-product inhibition, increased chemicalstability, improved chemoselectivity, improved solvent stability,increased tolerance to acidic pH, increased tolerance to proteolyticactivity (i.e., reduced sensitivity to proteolysis), reducedaggregation, increased solubility, and altered temperature profile. Inadditional embodiments, the term is used in reference to the at leastone improved property of uridine phosphorylase enzymes. In someembodiments, the present invention provides engineered uridinephosphorylase polypeptides that exhibit an improvement in any enzymeproperty as compared to a reference uridine phosphorylase polypeptideand/or a wild-type uridine phosphorylase polypeptide, and/or anotherengineered uridine phosphorylase polypeptide. Thus, the level of“improvement” can be determined and compared between various uridinephosphorylase polypeptides, including wild-type, as well as engineereduridine phosphorylases.

As used herein, “increased enzymatic activity” and “enhanced catalyticactivity” refer to an improved property of the engineered polypeptides,which can be represented by an increase in specific activity (e.g.,product produced/time/weight protein) or an increase in percentconversion of the substrate to the product (e.g., percent conversion ofstarting amount of substrate to product in a specified time period usinga specified amount of enzyme) as compared to the reference enzyme. Insome embodiments, the terms refer to an improved property of engineereduridine phosphorylase polypeptides provided herein, which can berepresented by an increase in specific activity (e.g., productproduced/time/weight protein) or an increase in percent conversion ofthe substrate to the product (e.g., percent conversion of startingamount of substrate to product in a specified time period using aspecified amount of uridine phosphorylase) as compared to the referenceuridine phosphorylase enzyme. In some embodiments, the terms are used inreference to improved uridine phosphorylase enzymes provided herein.Exemplary methods to determine enzyme activity of the engineered uridinephosphorylases of the present invention are provided in the Examples.Any property relating to enzyme activity may be affected, including theclassical enzyme properties of K_(m), V_(max) or k_(cat), changes ofwhich can lead to increased enzymatic activity. For example,improvements in enzyme activity can be from about 1.1 fold the enzymaticactivity of the corresponding wild-type enzyme, to as much as 2-fold,5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold,200-fold or more enzymatic activity than the naturally occurring uridinephosphorylase or another engineered uridine phosphorylase from which theuridine phosphorylase polypeptides were derived.

As used herein, “conversion” refers to the enzymatic conversion (orbiotransformation) of a substrate(s) to the corresponding product(s).“Percent conversion” refers to the percent of the substrate that isconverted to the product within a period of time under specifiedconditions. Thus, the “enzymatic activity” or “activity” of a uridinephosphorylase polypeptide can be expressed as “percent conversion” ofthe substrate to the product in a specific period of time.

Enzymes with “generalist properties” (or “generalist enzymes”) refer toenzymes that exhibit improved activity for a wide range of substrates,as compared to a parental sequence. Generalist enzymes do notnecessarily demonstrate improved activity for every possible substrate.In some embodiments, the present invention provides uridinephosphorylase variants with generalist properties, in that theydemonstrate similar or improved activity relative to the parental genefor a wide range of sterically and electronically diverse substrates. Inaddition, the generalist enzymes provided herein were engineered to beimproved across a wide range of diverse molecules to increase theproduction of metabolites/products.

The term “stringent hybridization conditions” is used herein to refer toconditions under which nucleic acid hybrids are stable. As known tothose of skill in the art, the stability of hybrids is reflected in themelting temperature (T_(m)) of the hybrids. In general, the stability ofa hybrid is a function of ion strength, temperature, G/C content, andthe presence of chaotropic agents. The T_(m) values for polynucleotidescan be calculated using known methods for predicting meltingtemperatures (See e.g., Baldino et al., Meth. Enzymol., 168:761-777[1989]; Bolton et al., Proc. Natl. Acad. Sci. USA 48:1390 [1962];Bresslauer et al., Proc. Natl. Acad. Sci. USA 83:8893-8897 [1986];Freier et al., Proc. Natl. Acad. Sci. USA 83:9373-9377 [1986]; Kierzeket al., Biochem., 25:7840-7846 [1986]; Rychlik et al., Nucl. Acids Res.,18:6409-6412 [1990] (erratum, Nucl. Acids Res., 19:698 [1991]); Sambrooket al., supra); Suggs et al., 1981, in Developmental Biology UsingPurified Genes, Brown et al. [eds.], pp. 683-693, Academic Press,Cambridge, Mass. [1981]; and Wetmur, Crit. Rev. Biochem. Mol. Biol.26:227-259 [1991]). In some embodiments, the polynucleotide encodes thepolypeptide disclosed herein and hybridizes under defined conditions,such as moderately stringent or highly stringent conditions, to thecomplement of a sequence encoding an engineered uridine phosphorylaseenzyme of the present invention.

As used herein, “hybridization stringency” relates to hybridizationconditions, such as washing conditions, in the hybridization of nucleicacids. Generally, hybridization reactions are performed under conditionsof lower stringency, followed by washes of varying but higherstringency. The term “moderately stringent hybridization” refers toconditions that permit target-DNA to bind a complementary nucleic acidthat has about 60% identity, preferably about 75% identity, about 85%identity to the target DNA, with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m), as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018 M NaCl at 65°C. (i.e., if a hybrid is not stable in 0.018 M NaCl at 65° C., it willnot be stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w/v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

As used herein, “codon optimized” refers to changes in the codons of thepolynucleotide encoding a protein to those preferentially used in aparticular organism such that the encoded protein is efficientlyexpressed in the organism of interest. Although the genetic code isdegenerate in that most amino acids are represented by several codons,called “synonyms” or “synonymous” codons, it is well known that codonusage by particular organisms is nonrandom and biased towards particularcodon triplets. This codon usage bias may be higher in reference to agiven gene, genes of common function or ancestral origin, highlyexpressed proteins versus low copy number proteins, and the aggregateprotein coding regions of an organism's genome. In some embodiments, thepolynucleotides encoding the uridine phosphorylase enzymes may be codonoptimized for optimal production in the host organism selected forexpression.

As used herein, “preferred,” “optimal,” and “high codon usage bias”codons when used alone or in combination refer(s) interchangeably tocodons that are used at higher frequency in the protein coding regionsthan other codons that code for the same amino acid. The preferredcodons may be determined in relation to codon usage in a single gene, aset of genes of common function or origin, highly expressed genes, thecodon frequency in the aggregate protein coding regions of the wholeorganism, codon frequency in the aggregate protein coding regions ofrelated organisms, or combinations thereof. Codons whose frequencyincreases with the level of gene expression are typically optimal codonsfor expression. A variety of methods are known for determining the codonfrequency (e.g., codon usage, relative synonymous codon usage) and codonpreference in specific organisms, including multivariate analysis, forexample, using cluster analysis or correspondence analysis, and theeffective number of codons used in a gene (See e.g., GCGCodonPreference, Genetics Computer Group Wisconsin Package; CodonW,Peden, University of Nottingham; McInerney, Bioinform., 14:372-73[1998]; Stenico et al., Nucl. Acids Res., 222437-46 [1994]; and Wright,Gene 87:23-29 [1990]). Codon usage tables are available for manydifferent organisms (See e.g., Wada et al., Nucl. Acids Res.,20:2111-2118 [1992]; Nakamura et al., Nucl. Acids Res., 28:292 [2000];Duret, et al., supra; Henaut and Danchin, in Escherichia coli andSalmonella, Neidhardt, et al. (eds.), ASM Press, Washington D.C., p.2047-2066 [1996]). The data source for obtaining codon usage may rely onany available nucleotide sequence capable of coding for a protein. Thesedata sets include nucleic acid sequences actually known to encodeexpressed proteins (e.g., complete protein coding sequences-CDS),expressed sequence tags (ESTS), or predicted coding regions of genomicsequences (See e.g., Mount, Bioinformatics: Sequence and GenomeAnalysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. [2001]; Uberbacher, Meth. Enzymol., 266:259-281 [1996]; andTiwari et al., Comput. Appl. Biosci., 13:263-270 [1997]).

As used herein, “control sequence” includes all components, which arenecessary or advantageous for the expression of a polynucleotide and/orpolypeptide of the present invention. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter sequence, signalpeptide sequence, initiation sequence and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleic acid sequence encoding a polypeptide.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide and/or polypeptide of interest.

“Promoter sequence” refers to a nucleic acid sequence that is recognizedby a host cell for expression of a polynucleotide of interest, such as acoding sequence. The promoter sequence contains transcriptional controlsequences, which mediate the expression of a polynucleotide of interest.The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

The phrase “suitable reaction conditions” refers to those conditions inthe enzymatic conversion reaction solution (e.g., ranges of enzymeloading, substrate loading, temperature, pH, buffers, co-solvents, etc.)under which a uridine phosphorylase polypeptide of the present inventionis capable of converting a substrate to the desired product compound.Some exemplary “suitable reaction conditions” are provided herein.

As used herein, “loading,” such as in “compound loading” or “enzymeloading” refers to the concentration or amount of a component in areaction mixture at the start of the reaction.

As used herein, “substrate” in the context of an enzymatic conversionreaction process refers to the compound or molecule acted on by theengineered enzymes provided herein (e.g., engineered uridinephosphorylase polypeptides).

As used herein, “increasing” yield of a product (e.g., a deoxyribosephosphate analogue) from a reaction occurs when a particular componentpresent during the reaction (e.g., a uridine phosphorylase enzyme)causes more product to be produced, compared with a reaction conductedunder the same conditions with the same substrate and othersubstituents, but in the absence of the component of interest.

A reaction is said to be “substantially free” of a particular enzyme ifthe amount of that enzyme compared with other enzymes that participatein catalyzing the reaction is less than about 2%, about 1%, or about0.1% (wt/wt).

As used herein, “fractionating” a liquid (e.g., a culture broth) meansapplying a separation process (e.g., salt precipitation, columnchromatography, size exclusion, and filtration) or a combination of suchprocesses to provide a solution in which a desired protein comprises agreater percentage of total protein in the solution than in the initialliquid product.

As used herein, “starting composition” refers to any composition thatcomprises at least one substrate. In some embodiments, the startingcomposition comprises any suitable substrate.

As used herein, “product” in the context of an enzymatic conversionprocess refers to the compound or molecule resulting from the action ofan enzymatic polypeptide on a substrate.

As used herein, “equilibration” as used herein refers to the processresulting in a steady state concentration of chemical species in achemical or enzymatic reaction (e.g., interconversion of two species Aand B), including interconversion of stereoisomers, as determined by theforward rate constant and the reverse rate constant of the chemical orenzymatic reaction.

As used herein, “alkyl” refers to saturated hydrocarbon groups of from 1to 18 carbon atoms inclusively, either straight chained or branched,more preferably from 1 to 8 carbon atoms inclusively, and mostpreferably 1 to 6 carbon atoms inclusively. An alkyl with a specifiednumber of carbon atoms is denoted in parenthesis (e.g., (C1-C4)alkylrefers to an alkyl of 1 to 4 carbon atoms).

As used herein, “alkenyl” refers to groups of from 2 to 12 carbon atomsinclusively, either straight or branched containing at least one doublebond but optionally containing more than one double bond.

As used herein, “alkynyl” refers to groups of from 2 to 12 carbon atomsinclusively, either straight or branched containing at least one triplebond but optionally containing more than one triple bond, andadditionally optionally containing one or more double bonded moieties.

As used herein, “heteroalkyl, “heteroalkenyl,” and heteroalkynyl,” referto alkyl, alkenyl and alkynyl as defined herein in which one or more ofthe carbon atoms are each independently replaced with the same ordifferent heteroatoms or heteroatomic groups. Heteroatoms and/orheteroatomic groups which can replace the carbon atoms include, but arenot limited to, —O—, —S—, —S—O—, —NRα—, —PH—, —S(O)—, —S(O)2—,—S(O)—NRα—, —S(O)2NRα—, and the like, including combinations thereof,where each Ra is independently selected from hydrogen, alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

As used herein, “alkoxy” refers to the group —ORβ wherein Rβ is an alkylgroup is as defined above including optionally substituted alkyl groupsas also defined herein.

As used herein, “aryl” refers to an unsaturated aromatic carbocyclicgroup of from 6 to 12 carbon atoms inclusively having a single ring(e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl).Exemplary aryls include phenyl, pyridyl, naphthyl and the like.

As used herein, “amino” refers to the group —NH2. Substituted aminorefers to the group —NHRδ, NRδRδ, and NRδRδRδ, where each Rδ isindependently selected from substituted or unsubstituted alkyl,cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl,acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like.Typical amino groups include, but are limited to, dimethylamino,diethylamino, trimethylammonium, triethylammonium, methylysulfonylamino,furanyl-oxy-sulfamino, and the like.

As used herein, “oxo” refers to ═O.

As used herein, “oxy” refers to a divalent group —O—, which may havevarious substituents to form different oxy groups, including ethers andesters.

As used herein, “carboxy” refers to —COOH.

As used herein, “carbonyl” refers to —C(O)—, which may have a variety ofsubstituents to form different carbonyl groups including acids, acidhalides, aldehydes, amides, esters, and ketones.

As used herein, “alkyloxycarbonyl” refers to —C(O)ORϵ, where Rϵ is analkyl group as defined herein, which can be optionally substituted.

As used herein, “aminocarbonyl” refers to —C(O)NH2. Substitutedaminocarbonyl refers to —C(O)NRδRδ, where the amino group NRδRδ is asdefined herein.

As used herein, “halogen” and “halo” refer to fluoro, chloro, bromo andiodo.

As used herein, “hydroxy” refers to —OH.

As used herein, “cyano” refers to —CN.

As used herein, “heteroaryl” refers to an aromatic heterocyclic group offrom 1 to 10 carbon atoms inclusively and 1 to 4 heteroatoms inclusivelyselected from oxygen, nitrogen and sulfur within the ring. Suchheteroaryl groups can have a single ring (e.g., pyridyl or furyl) ormultiple condensed rings (e.g., indolizinyl or benzothienyl).

As used herein, “heteroarylalkyl” refers to an alkyl substituted with aheteroaryl (i.e., heteroaryl-alkyl-groups), preferably having from 1 to6 carbon atoms inclusively in the alkyl moiety and from 5 to 12 ringatoms inclusively in the heteroaryl moiety. Such heteroarylalkyl groupsare exemplified by pyridylmethyl and the like.

As used herein, “heteroarylalkenyl” refers to an alkenyl substitutedwith a heteroaryl (i.e., heteroaryl-alkenyl-groups), preferably havingfrom 2 to 6 carbon atoms inclusively in the alkenyl moiety and from 5 to12 ring atoms inclusively in the heteroaryl moiety.

As used herein, “heteroarylalkynyl” refers to an alkynyl substitutedwith a heteroaryl (i.e., heteroaryl-alkynyl-groups), preferably havingfrom 2 to 6 carbon atoms inclusively in the alkynyl moiety and from 5 to12 ring atoms inclusively in the heteroaryl moiety.

As used herein, “heterocycle,” “heterocyclic,” and interchangeably“heterocycloalkyl,” refer to a saturated or unsaturated group having asingle ring or multiple condensed rings, from 2 to 10 carbon ring atomsinclusively and from 1 to 4 hetero ring atoms inclusively selected fromnitrogen, sulfur or oxygen within the ring. Such heterocyclic groups canhave a single ring (e.g., piperidinyl or tetrahydrofuryl) or multiplecondensed rings (e.g., indolinyl, dihydrobenzofuran or quinuclidinyl).Examples of heterocycles include, but are not limited to, furan,thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine,pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, pyrrolidine,indoline and the like.

As used herein, “membered ring” is meant to embrace any cyclicstructure. The number preceding the term “membered” denotes the numberof skeletal atoms that constitute the ring. Thus, for example,cyclohexyl, pyridine, pyran and thiopyran are 6-membered rings andcyclopentyl, pyrrole, furan, and thiophene are 5-membered rings.

Unless otherwise specified, positions occupied by hydrogen in theforegoing groups can be further substituted with substituentsexemplified by, but not limited to, hydroxy, oxo, nitro, methoxy,ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy,fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl,alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl,hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxy,alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl,alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido,cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl,acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substitutedaryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl,heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl,heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl,substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl,morpholino, heterocycle, (heterocycle)oxy, and (heterocycle)alkyl; andpreferred heteroatoms are oxygen, nitrogen, and sulfur. It is understoodthat where open valences exist on these substituents they can be furthersubstituted with alkyl, cycloalkyl, aryl, heteroaryl, and/or heterocyclegroups, that where these open valences exist on carbon they can befurther substituted by halogen and by oxygen-, nitrogen-, orsulfur-bonded substituents, and where multiple such open valences exist,these groups can be joined to form a ring, either by direct formation ofa bond or by formation of bonds to a new heteroatom, preferably oxygen,nitrogen, or sulfur. It is further understood that the abovesubstitutions can be made provided that replacing the hydrogen with thesubstituent does not introduce unacceptable instability to the moleculesof the present invention, and is otherwise chemically reasonable.

As used herein the term “culturing” refers to the growing of apopulation of microbial cells under any suitable conditions (e.g., usinga liquid, gel or solid medium).

Recombinant polypeptides can be produced using any suitable methodsknown in the art. Genes encoding the wild-type polypeptide of interestcan be cloned in vectors, such as plasmids, and expressed in desiredhosts, such as E. coli, etc. Variants of recombinant polypeptides can begenerated by various methods known in the art. Indeed, there is a widevariety of different mutagenesis techniques well known to those skilledin the art. In addition, mutagenesis kits are also available from manycommercial molecular biology suppliers. Methods are available to makespecific substitutions at defined amino acids (site-directed), specificor random mutations in a localized region of the gene (regio-specific),or random mutagenesis over the entire gene (e.g., saturationmutagenesis). Numerous suitable methods are known to those in the art togenerate enzyme variants, including but not limited to site-directedmutagenesis of single-stranded DNA or double-stranded DNA using PCR,cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, andchemical saturation mutagenesis, or any other suitable method known inthe art. Mutagenesis and directed evolution methods can be readilyapplied to enzyme-encoding polynucleotides to generate variant librariesthat can be expressed, screened, and assayed. Any suitable mutagenesisand directed evolution methods find use in the present invention and arewell known in the art (See e.g., U.S. Pat. Nos. 5,605,793, 5,811,238,5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679,6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201, 6,277,638,6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883,6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198,6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377,6,365,408, 6,368,861, 6,372,497, 6,337,186, 6,376,246, 6,379,964,6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910,6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675,6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011, 6,484,105,6,489,146, 6,500,617, 6,500,639, 6,506,602, 6,506,603, 6,518,065,6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467,6,579,678, 6,586,182, 6,602,986, 6,605,430, 6,613,514, 6,653,072,6,686,515, 6,703,240, 6,716,631, 6,825,001, 6,902,922, 6,917,882,6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297,7,148,054, 7,220,566, 7,288,375, 7,384,387, 7,421,347, 7,430,477,7,462,469, 7,534,564, 7,620,500, 7,620,502, 7,629,170, 7,702,464,7,747,391, 7,747,393, 7,751,986, 7,776,598, 7,783,428, 7,795,030,7,853,410, 7,868,138, 7,783,428, 7,873,477, 7,873,499, 7,904,249,7,957,912, 7,981,614, 8,014,961, 8,029,988, 8,048,674, 8,058,001,8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346,8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326,9,665,694, 9,684,771, and all related US, as well as PCT and non-UScounterparts; Ling et al., Anal. Biochem., 254(2):157-78 [1997]; Dale etal., Meth. Mol. Biol., 57:369-74 [1996]; Smith, Ann. Rev. Genet.,19:423-462 [1985]; Botstein et al., Science, 229:1193-1201 [1985];Carter, Biochem. J., 237:1-7 [1986]; Kramer et al., Cell, 38:879-887[1984]; Wells et al., Gene, 34:315-323 [1985]; Minshull et al., Curr.Op. Chem. Biol., 3:284-290 [1999]; Christians et al., Nat. Biotechnol.,17:259-264 [1999]; Crameri et al., Nature, 391:288-291 [1998]; Crameri,et al., Nat. Biotechnol., 15:436-438 [1997]; Zhang et al., Proc. Nat.Acad. Sci. U.S.A., 94:4504-4509 [1997]; Crameri et al., Nat.Biotechnol., 14:315-319 [1996]; Stemmer, Nature, 370:389-391 [1994];Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751 [1994]; WO 95/22625;WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767; and WO2009/152336, all of which are incorporated herein by reference).

In some embodiments, the enzyme clones obtained following mutagenesistreatment are screened by subjecting the enzyme preparations to adefined temperature (or other assay conditions) and measuring the amountof enzyme activity remaining after heat treatments or other suitableassay conditions. Clones containing a polynucleotide encoding apolypeptide are then isolated from the gene, sequenced to identify thenucleotide sequence changes (if any), and used to express the enzyme ina host cell. Measuring enzyme activity from the expression libraries canbe performed using any suitable method known in the art (e.g., standardbiochemistry techniques, such as HPLC analysis).

After the variants are produced, they can be screened for any desiredproperty (e.g., high or increased activity, or low or reduced activity,increased thermal activity, increased thermal stability, and/or acidicpH stability, etc.). In some embodiments, “recombinant uridinephosphorylase polypeptides” (also referred to herein as “engineereduridine phosphorylase polypeptides,” “variant uridine phosphorylaseenzymes,” “uridine phosphorylase variants,” and “uridine phosphorylasecombinatorial variants”) find use.

As used herein, a “vector” is a DNA construct for introducing a DNAsequence into a cell. In some embodiments, the vector is an expressionvector that is operably linked to a suitable control sequence capable ofeffecting the expression in a suitable host of the polypeptide encodedin the DNA sequence. In some embodiments, an “expression vector” has apromoter sequence operably linked to the DNA sequence (e.g., transgene)to drive expression in a host cell, and in some embodiments, alsocomprises a transcription terminator sequence.

As used herein, the term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation, andpost-translational modification. In some embodiments, the term alsoencompasses secretion of the polypeptide from a cell.

As used herein, the term “produces” refers to the production of proteinsand/or other compounds by cells. It is intended that the term encompassany step involved in the production of polypeptides including, but notlimited to, transcription, post-transcriptional modification,translation, and post-translational modification. In some embodiments,the term also encompasses secretion of the polypeptide from a cell.

As used herein, an amino acid or nucleotide sequence (e.g., a promotersequence, signal peptide, terminator sequence, etc.) is “heterologous”to another sequence with which it is operably linked if the twosequences are not associated in nature. For example, a “heterologouspolynucleotide” is any polynucleotide that is introduced into a hostcell by laboratory techniques, and includes polynucleotides that areremoved from a host cell, subjected to laboratory manipulation, and thenreintroduced into a host cell.

As used herein, the terms “host cell” and “host strain” refer tosuitable hosts for expression vectors comprising DNA provided herein(e.g., the polynucleotides encoding the uridine phosphorylase variants).In some embodiments, the host cells are prokaryotic or eukaryotic cellsthat have been transformed or transfected with vectors constructed usingrecombinant DNA techniques as known in the art.

The term “analogue” means a polypeptide having more than 70% sequenceidentity but less than 100% sequence identity (e.g., more than 75%, 78%,80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity) with a reference polypeptide. In some embodiments,analogues mean polypeptides that contain one or more non-naturallyoccurring amino acid residues including, but not limited, tohomoarginine, ornithine and norvaline, as well as naturally occurringamino acids. In some embodiments, analogs also include one or moreD-amino acid residues and non-peptide linkages between two or more aminoacid residues.

The term “effective amount” means an amount sufficient to produce thedesired result. One of general skill in the art may determine what theeffective amount by using routine experimentation.

The terms “isolated” and “purified” are used to refer to a molecule(e.g., an isolated nucleic acid, polypeptide, etc.) or other componentthat is removed from at least one other component with which it isnaturally associated. The term “purified” does not require absolutepurity, rather it is intended as a relative definition.

As used herein, “stereoselectivity” refers to the preferential formationin a chemical or enzymatic reaction of one stereoisomer over another.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (“e.e.”) calculated therefromaccording to the formula [major enantiomer−minor enantiomer]/[majorenantiomer+minor enantiomer]. Where the stereoisomers arediastereoisomers, the stereoselectivity is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diastereomers, commonlyalternatively reported as the diastereomeric excess (“d.e.”).Enantiomeric excess and diastereomeric excess are types of stereomericexcess.

As used herein, “regioselectivity” and “regioselective reaction” referto a reaction in which one direction of bond making or breaking occurspreferentially over all other possible directions. Reactions cancompletely (100%) regioselective if the discrimination is complete,substantially regioselective (at least 75%), or partially regioselective(x %, wherein the percentage is set dependent upon the reaction ofinterest), if the product of reaction at one site predominates over theproduct of reaction at other sites.

As used herein, “chemoselectivity” refers to the preferential formationin a chemical or enzymatic reaction of one product over another.

As used herein, “pH stable” refers to a uridine phosphorylasepolypeptide that maintains similar activity (e.g., more than 60% to 80%)after exposure to high or low pH (e.g., 4.5-6 or 8 to 12) for a periodof time (e.g., 0.5-24 hrs) compared to the untreated enzyme.

As used herein, “thermostable” refers to a uridine phosphorylasepolypeptide that maintains similar activity (more than 60% to 80% forexample) after exposure to elevated temperatures (e.g., 40-80° C.) for aperiod of time (e.g., 0.5-24 h) compared to the wild-type enzyme exposedto the same elevated temperature.

As used herein, “solvent stable” refers to a uridine phosphorylasepolypeptide that maintains similar activity (more than e.g., 60% to 80%)after exposure to varying concentrations (e.g., 5-99%) of solvent(ethanol, isopropyl alcohol, dimethylsulfoxide [DMSO], tetrahydrofuran,2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyltert-butyl ether, etc.) for a period of time (e.g., 0.5-24 h) comparedto the wild-type enzyme exposed to the same concentration of the samesolvent.

As used herein, “thermo- and solvent stable” refers to a uridinephosphorylase polypeptide that is both thermostable and solvent stable.

As used herein, “optional” and “optionally” mean that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. One of ordinary skill in the artwould understand that with respect to any molecule described ascontaining one or more optional substituents, only sterically practicaland/or synthetically feasible compounds are meant to be included.

As used herein, “optionally substituted” refers to all subsequentmodifiers in a term or series of chemical groups. For example, in theterm “optionally substituted arylalkyl, the “alkyl” portion and the“aryl” portion of the molecule may or may not be substituted, and forthe series “optionally substituted alkyl, cycloalkyl, aryl andheteroaryl,” the alkyl, cycloalkyl, aryl, and heteroaryl groups,independently of the others, may or may not be substituted.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides engineered uridine phosphorylase (UP)enzymes, polypeptides having UP activity, and polynucleotides encodingthese enzymes, as well as vectors and host cells comprising thesepolynucleotides and polypeptides. Methods for producing UP enzymes arealso provided. The present invention further provides compositionscomprising the UP enzymes and methods of using the engineered UPenzymes. The present invention finds particular use in the production ofpharmaceutical compounds.

Increasingly, non-natural nucleoside analogs are being investigated forthe treatment of cancer and viral infections, such as COVID-19.Industrial process conditions often require the efficient production ofnon-natural nucleosides using intermediates and substrates that arereadily available and cost effective. One such intermediate is compound(1), 5′-isobutyryluridine.

There is a need in the art for methods, more particularly greenchemistry methods, to synthesize compound (1) under industrial processconditions. One such method is to use a biocatalyst or engineered enzymeto produce compound (1). In some embodiments, this disclosure providesengineered uridine phosphorylase enzymes for the production of compound(1). The engineered uridine phosphorylase enzymes of the presentdisclosure generate compound (1) from the substrates of 5′-isobutyrylribose-1-phosphate, compound (2) and uracil, compound (3). See Scheme I,below.

Inorganic phosphate (not pictured) is also generated in the conversionof compound (2) and compound (3) to compound (1). Addition of sucrosephosphorylase (or any one of a number of enzymes that consume inorganicphosphate, e.g. pyruvate oxidase) is used to drive the equilibrium ofthe reversible reaction toward the product of compound (1).

Engineered uridine phosphorylase enzymes with improved propertiescompared to naturally occurring uridine phosphorylases may be used underrelevant process conditions and/or in multi-enzyme systems. Theseengineered UP enzymes may result in improved production of compound (1)and/or may have other improved properties.

There is a need for engineered UPs that have improved activity and thatoperate under typical industrial conditions and/or as part ofmulti-enzyme systems. The present invention addresses this need andprovides engineered UPs that are suitable for use in these and otherreactions under industrial conditions.

Engineered UP Polypeptides

The present invention provides engineered UP polypeptides,polynucleotides encoding the polypeptides, methods of preparing thepolypeptides, and methods for using the polypeptides. Where thedescription relates to polypeptides, it is to be understood that it alsodescribes the polynucleotides encoding the polypeptides. In someembodiments, the present invention provides engineered, non-naturallyoccurring UP enzymes with improved properties as compared to wild-typeUP enzymes. Any suitable reaction conditions find use in the presentinvention. In some embodiments, methods are used to analyze the improvedproperties of the engineered polypeptides to carry out thephosphorylation reaction. In some embodiments, the reaction conditionsare modified with regard to concentrations or amounts of engineered UP,substrate(s), buffer(s), solvent(s), pH, conditions includingtemperature and reaction time, and/or conditions with the engineered UPpolypeptide immobilized on a solid support, as further described belowand in the Examples.

In some embodiments, additional reaction components or additionaltechniques are utilized to supplement the reaction conditions. In someembodiments, these include taking measures to stabilize or preventinactivation of the enzyme, reduce product inhibition, shift reactionequilibrium to desired product formation.

In some further embodiments, any of the above described process for theconversion of substrate compound to product compound can furthercomprise one or more steps selected from: extraction, isolation,purification, crystallization, filtration, and/or lyophilization ofproduct compound(s). Methods, techniques, and protocols for extracting,isolating, purifying, and/or crystallizing the product(s) frombiocatalytic reaction mixtures produced by the processes provided hereinare known to the ordinary artisan and/or accessed through routineexperimentation. Additionally, illustrative methods are provided in theExamples below.

Engineered UP Polynucleotides Encoding Engineered Polypeptides,Expression Vectors and Host Cells

The present invention provides polynucleotides encoding the engineeredenzyme polypeptides described herein. In some embodiments, thepolynucleotides are operatively linked to one or more heterologousregulatory sequences that control gene expression to create arecombinant polynucleotide capable of expressing the polypeptide. Insome embodiments, expression constructs containing at least oneheterologous polynucleotide encoding the engineered enzymepolypeptide(s) is introduced into appropriate host cells to express thecorresponding enzyme polypeptide(s).

As will be apparent to the skilled artisan, availability of a proteinsequence and the knowledge of the codons corresponding to the variousamino acids provide a description of all the polynucleotides capable ofencoding the subject polypeptides. The degeneracy of the genetic code,where the same amino acids are encoded by alternative or synonymouscodons, allows an extremely large number of nucleic acids to be made,all of which encode an engineered enzyme (e.g., UP) polypeptide. Thus,the present invention provides methods and compositions for theproduction of each and every possible variation of enzymepolynucleotides that could be made that encode the enzyme polypeptidesdescribed herein by selecting combinations based on the possible codonchoices, and all such variations are to be considered specificallydisclosed for any polypeptide described herein, including the amino acidsequences presented in the Examples (e.g., in the various Tables).

In some embodiments, the codons are preferably optimized for utilizationby the chosen host cell for protein production. For example, preferredcodons used in bacteria are typically used for expression in bacteria.Consequently, codon optimized polynucleotides encoding the engineeredenzyme polypeptides contain preferred codons at about 40%, 50%, 60%,70%, 80%, 90%, or greater than 90% of the codon positions in the fulllength coding region.

In some embodiments, the enzyme polynucleotide encodes an engineeredpolypeptide having enzyme activity with the properties disclosed herein,wherein the polypeptide comprises an amino acid sequence having at least60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequenceselected from the SEQ ID NOs provided herein, or the amino acid sequenceof any variant (e.g., those provided in the Examples), and one or moreresidue differences as compared to the reference polynucleotide(s), orthe amino acid sequence of any variant as disclosed in the Examples (forexample 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residuepositions). In some embodiments, the reference polypeptide sequence isselected from SEQ ID NOs: 2, 4, 246, 594, 776, and/or 868.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a reference polynucleotide sequenceselected from any polynucleotide sequence provided herein, or acomplement thereof, or a polynucleotide sequence encoding any of thevariant enzyme polypeptides provided herein. In some embodiments, thepolynucleotide capable of hybridizing under highly stringent conditionsencodes an enzyme polypeptide comprising an amino acid sequence that hasone or more residue differences as compared to a reference sequence.

In some embodiments, an isolated polynucleotide encoding any of theengineered enzyme polypeptides herein is manipulated in a variety ofways to facilitate expression of the enzyme polypeptide. In someembodiments, the polynucleotides encoding the enzyme polypeptidescomprise expression vectors where one or more control sequences ispresent to regulate the expression of the enzyme polynucleotides and/orpolypeptides. Manipulation of the isolated polynucleotide prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector utilized. Techniques for modifying polynucleotides andnucleic acid sequences utilizing recombinant DNA methods are well knownin the art. In some embodiments, the control sequences include amongothers, promoters, leader sequences, polyadenylation sequences,propeptide sequences, signal peptide sequences, and transcriptionterminators. In some embodiments, suitable promoters are selected basedon the host cells selection. For bacterial host cells, suitablepromoters for directing transcription of the nucleic acid constructs ofthe present disclosure, include, but are not limited to promotersobtained from the E. coli lac operon, Streptomyces coelicolor agarasegene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacilluslicheniformis alpha-amylase gene (amyL), Bacillus stearothermophilusmaltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylasegene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillussubtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (Seee.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75: 3727-3731[1978]), as well as the tac promoter (See e.g., DeBoer et al., Proc.Natl Acad. Sci. USA 80: 21-25 [1983]). Exemplary promoters forfilamentous fungal host cells, include, but are not limited to promotersobtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucormiehei aspartic proteinase, Aspergillus niger neutral alpha-amylase,Aspergillus niger acid stable alpha-amylase, Aspergillus niger orAspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase,Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusariumoxysporum trypsin-like protease (See e.g., WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof. Exemplary yeast cell promoters can be from the genes can befrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase(ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.Other useful promoters for yeast host cells are known in the art (Seee.g., Romanos et al., Yeast 8:423-488 [1992]).

In some embodiments, the control sequence is also a suitabletranscription terminator sequence (i.e., a sequence recognized by a hostcell to terminate transcription). In some embodiments, the terminatorsequence is operably linked to the 3′ terminus of the nucleic acidsequence encoding the enzyme polypeptide. Any suitable terminator whichis functional in the host cell of choice finds use in the presentinvention. Exemplary transcription terminators for filamentous fungalhost cells can be obtained from the genes for Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease. Exemplary terminators for yeast hostcells can be obtained from the genes for Saccharomyces cerevisiaeenolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomycescerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other usefulterminators for yeast host cells are known in the art (See e.g., Romanoset al., supra).

In some embodiments, the control sequence is also a suitable leadersequence (i.e., a non-translated region of an mRNA that is important fortranslation by the host cell). In some embodiments, the leader sequenceis operably linked to the 5′ terminus of the nucleic acid sequenceencoding the enzyme polypeptide. Any suitable leader sequence that isfunctional in the host cell of choice find use in the present invention.Exemplary leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulanstriose phosphate isomerase. Suitable leaders for yeast host cells areobtained from the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

In some embodiments, the control sequence is also a polyadenylationsequence (i.e., a sequence operably linked to the 3′ terminus of thenucleic acid sequence and which, when transcribed, is recognized by thehost cell as a signal to add polyadenosine residues to transcribedmRNA). Any suitable polyadenylation sequence which is functional in thehost cell of choice finds use in the present invention. Exemplarypolyadenylation sequences for filamentous fungal host cells include, butare not limited to the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase. Useful polyadenylation sequences for yeast hostcells are known (See e.g., Guo and Sherman, Mol. Cell. Bio.,15:5983-5990 [1995]).

In some embodiments, the control sequence is also a signal peptide(i.e., a coding region that codes for an amino acid sequence linked tothe amino terminus of a polypeptide and directs the encoded polypeptideinto the cell's secretory pathway). In some embodiments, the 5′ end ofthe coding sequence of the nucleic acid sequence inherently contains asignal peptide coding region naturally linked in translation readingframe with the segment of the coding region that encodes the secretedpolypeptide. Alternatively, in some embodiments, the 5′ end of thecoding sequence contains a signal peptide coding region that is foreignto the coding sequence. Any suitable signal peptide coding region whichdirects the expressed polypeptide into the secretory pathway of a hostcell of choice finds use for expression of the engineeredpolypeptide(s). Effective signal peptide coding regions for bacterialhost cells are the signal peptide coding regions include, but are notlimited to those obtained from the genes for Bacillus NClB 11837maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacilluslicheniformis subtilisin, Bacillus licheniformis beta-lactamase,Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), andBacillus subtilis prsA. Further signal peptides are known in the art(See e.g., Simonen and Palva, Microbiol. Rev., 57:109-137 [1993]). Insome embodiments, effective signal peptide coding regions forfilamentous fungal host cells include, but are not limited to the signalpeptide coding regions obtained from the genes for Aspergillus oryzaeTAKA amylase, Aspergillus niger neutral amylase, Aspergillus nigerglucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolenscellulase, and Humicola lanuginosa lipase. Useful signal peptides foryeast host cells include, but are not limited to those from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase.

In some embodiments, the control sequence is also a propeptide codingregion that codes for an amino acid sequence positioned at the aminoterminus of a polypeptide. The resultant polypeptide is referred to as a“proenzyme,” “propolypeptide,” or “zymogen.” A propolypeptide can beconverted to a mature active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding region may be obtained from any suitable source, including, butnot limited to the genes for Bacillus subtilis alkaline protease (aprE),Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiaealpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthorathermophila lactase (See e.g., WO 95/33836). Where both signal peptideand propeptide regions are present at the amino terminus of apolypeptide, the propeptide region is positioned next to the aminoterminus of a polypeptide and the signal peptide region is positionednext to the amino terminus of the propeptide region.

In some embodiments, regulatory sequences are also utilized. Thesesequences facilitate the regulation of the expression of the polypeptiderelative to the growth of the host cell. Examples of regulatory systemsare those that cause the expression of the gene to be turned on or offin response to a chemical or physical stimulus, including the presenceof a regulatory compound. In prokaryotic host cells, suitable regulatorysequences include, but are not limited to the lac, tac, and trp operatorsystems. In yeast host cells, suitable regulatory systems include, butare not limited to the ADH2 system or GAL1 system. In filamentous fungi,suitable regulatory sequences include, but are not limited to the TAKAalpha-amylase promoter, Aspergillus niger glucoamylase promoter, andAspergillus oryzae glucoamylase promoter.

In another aspect, the present invention is directed to a recombinantexpression vector comprising a polynucleotide encoding an engineeredenzyme polypeptide, and one or more expression regulating regions suchas a promoter and a terminator, a replication origin, etc., depending onthe type of hosts into which they are to be introduced. In someembodiments, the various nucleic acid and control sequences describedherein are joined together to produce recombinant expression vectorswhich include one or more convenient restriction sites to allow forinsertion or substitution of the nucleic acid sequence encoding theenzyme polypeptide at such sites. Alternatively, in some embodiments,the nucleic acid sequence of the present invention is expressed byinserting the nucleic acid sequence or a nucleic acid constructcomprising the sequence into an appropriate vector for expression. Insome embodiments involving the creation of the expression vector, thecoding sequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any suitable vector (e.g., aplasmid or virus), that can be conveniently subjected to recombinant DNAprocedures and bring about the expression of the enzyme polynucleotidesequence. The choice of the vector typically depends on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

In some embodiments, the expression vector is an autonomouslyreplicating vector (i.e., a vector that exists as an extra-chromosomalentity, the replication of which is independent of chromosomalreplication, such as a plasmid, an extra-chromosomal element, aminichromosome, or an artificial chromosome). The vector may contain anymeans for assuring self-replication. In some alternative embodiments,the vector is one in which, when introduced into the host cell, it isintegrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, in someembodiments, a single vector or plasmid, or two or more vectors orplasmids which together contain the total DNA to be introduced into thegenome of the host cell, and/or a transposon is utilized.

In some embodiments, the expression vector contains one or moreselectable markers, which permit easy selection of transformed cells. A“selectable marker” is a gene, the product of which provides for biocideor viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markersinclude, but are not limited to the dal genes from Bacillus subtilis orBacillus licheniformis, or markers, which confer antibiotic resistancesuch as ampicillin, kanamycin, chloramphenicol or tetracyclineresistance. Suitable markers for yeast host cells include, but are notlimited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectablemarkers for use in filamentous fungal host cells include, but are notlimited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae),argB (ornithine carbamoyltransferases), bar (phosphinothricinacetyltransferase; e.g., from S. hygroscopicus), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase; e.g., from A. nidulans or A.orzyae), sC (sulfate adenyltransferase), and trpC (anthranilatesynthase), as well as equivalents thereof.

In another aspect, the present invention provides a host cell comprisingat least one polynucleotide encoding at least one engineered enzymepolypeptide of the present invention, the polynucleotide(s) beingoperatively linked to one or more control sequences for expression ofthe engineered enzyme enzyme(s) in the host cell. Host cells suitablefor use in expressing the polypeptides encoded by the expression vectorsof the present invention are well known in the art and include but arenot limited to, bacterial cells, such as E. coli, Vibrio fluvialis,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowesmelanoma cells; and plant cells. Exemplary host cells also includevarious Escherichia coli strains (e.g., W3110 (ΔfhuA) and BL21).Examples of bacterial selectable markers include, but are not limited tothe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers, which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol, and or tetracycline resistance.

In some embodiments, the expression vectors of the present inventioncontain an element(s) that permits integration of the vector into thehost cell's genome or autonomous replication of the vector in the cellindependent of the genome. In some embodiments involving integrationinto the host cell genome, the vectors rely on the nucleic acid sequenceencoding the polypeptide or any other element of the vector forintegration of the vector into the genome by homologous or nonhomologousrecombination.

In some alternative embodiments, the expression vectors containadditional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements preferably contain a sufficient number ofnucleotides, such as 100 to 10,000 base pairs, preferably 400 to 10,000base pairs, and most preferably 800 to 10,000 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are P15Aori or the origins of replication of plasmids pBR322, pUC19, pACYC177(which plasmid has the P15A ori), or pACYC184 permitting replication inE. coli, and pUB110, pE194, or pTA1060 permitting replication inBacillus. Examples of origins of replication for use in a yeast hostcell are the 2 micron origin of replication, ARS1, ARS4, the combinationof ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin ofreplication may be one having a mutation which makes its functioningtemperature-sensitive in the host cell (See e.g., Ehrlich, Proc. Natl.Acad. Sci. USA 75:1433 [1978]).

In some embodiments, more than one copy of a nucleic acid sequence ofthe present invention is inserted into the host cell to increaseproduction of the gene product. An increase in the copy number of thenucleic acid sequence can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome or byincluding an amplifiable selectable marker gene with the nucleic acidsequence where cells containing amplified copies of the selectablemarker gene, and thereby additional copies of the nucleic acid sequence,can be selected for by cultivating the cells in the presence of theappropriate selectable agent.

Many of the expression vectors for use in the present invention arecommercially available. Suitable commercial expression vectors include,but are not limited to the p3xFLAGTM™ expression vectors (Sigma-AldrichChemicals), which include a CMV promoter and hGH polyadenylation sitefor expression in mammalian host cells and a pBR322 origin ofreplication and ampicillin resistance markers for amplification in E.coli. Other suitable expression vectors include, but are not limited topBluescriptII SK(−) and pBK-CMV (Stratagene), and plasmids derived frompBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly(See e.g., Lathe et al., Gene 57:193-201 [1987]).

Thus, in some embodiments, a vector comprising a sequence encoding atleast one variant uridine phosphorylase is transformed into a host cellin order to allow propagation of the vector and expression of thevariant uridine phosphorylase(s). In some embodiments, the varianturidine phosphorylases are post-translationally modified to remove thesignal peptide, and in some cases, may be cleaved after secretion. Insome embodiments, the transformed host cell described above is culturedin a suitable nutrient medium under conditions permitting the expressionof the variant uridine phosphorylase(s). Any suitable medium useful forculturing the host cells finds use in the present invention, including,but not limited to minimal or complex media containing appropriatesupplements. In some embodiments, host cells are grown in HTP media.Suitable media are available from various commercial suppliers or may beprepared according to published recipes (e.g., in catalogues of theAmerican Type Culture Collection).

In another aspect, the present invention provides host cells comprisinga polynucleotide encoding an improved uridine phosphorylase polypeptideprovided herein, the polynucleotide being operatively linked to one ormore control sequences for expression of the uridine phosphorylaseenzyme in the host cell. Host cells for use in expressing the uridinephosphorylase polypeptides encoded by the expression vectors of thepresent invention are well known in the art and include but are notlimited to, bacterial cells, such as E. coli, Bacillus megaterium,Lactobacillus kefir, Streptomyces and Salmonella typhimurium cells;fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae orPichia pastoris (ATCC Accession No. 201178)); insect cells such asDrosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS,BHK, 293, and Bowes melanoma cells; and plant cells. Appropriate culturemedia and growth conditions for the above-described host cells are wellknown in the art.

Polynucleotides for expression of the uridine phosphorylase may beintroduced into cells by various methods known in the art. Techniquesinclude among others, electroporation, biolistic particle bombardment,liposome mediated transfection, calcium chloride transfection, andprotoplast fusion. Various methods for introducing polynucleotides intocells are known to those skilled in the art.

In some embodiments, the host cell is a eukaryotic cell. Suitableeukaryotic host cells include, but are not limited to, fungal cells,algal cells, insect cells, and plant cells. Suitable fungal host cellsinclude, but are not limited to, Ascomycota, Basidiomycota,Deuteromycota, Zygomycota, Fungi imperfecti. In some embodiments, thefungal host cells are yeast cells and filamentous fungal cells. Thefilamentous fungal host cells of the present invention include allfilamentous forms of the subdivision Eumycotina and Oomycota.Filamentous fungi are characterized by a vegetative mycelium with a cellwall composed of chitin, cellulose and other complex polysaccharides.The filamentous fungal host cells of the present invention aremorphologically distinct from yeast.

In some embodiments of the present invention, the filamentous fungalhost cells are of any suitable genus and species, including, but notlimited to Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera,Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus,Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis,Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora,Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces,Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium,Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes,Tolypocladium, Trichoderma, Verticillium, and/or Volvariella, and/orteleomorphs, or anamorphs, and synonyms, basionyms, or taxonomicequivalents thereof.

In some embodiments of the present invention, the host cell is a yeastcell, including but not limited to cells of Candida, Hansenula,Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, or Yarrowiaspecies. In some embodiments of the present invention, the yeast cell isHansenula polymorpha, Saccharomyces cerevisiae, Saccharomycescarlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis,Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris,Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichiamethanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, orYarrowia lipolytica.

In some embodiments of the invention, the host cell is an algal cellsuch as Chlamydomonas (e.g., C. reinhardtii) and Phormidium (P. sp.ATCC29409).

In some other embodiments, the host cell is a prokaryotic cell. Suitableprokaryotic cells include, but are not limited to Gram-positive,Gram-negative and Gram-variable bacterial cells. Any suitable bacterialorganism finds use in the present invention, including but not limitedto Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter,Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium,Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter,Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia,Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium,Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter,Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus,Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium,Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus,Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia,Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus,Synecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella,Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula,Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella,Yersinia and Zymomonas. In some embodiments, the host cell is a speciesof Agrobacterium, Acinetobacter, Azobacter, Bacillus, Bifidobacterium,Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium,Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus,Lactococcus, Pantoea, Pseudomonas, Staphylococcus, Salmonella,Streptococcus, Streptomyces, or Zymomonas. In some embodiments, thebacterial host strain is non-pathogenic to humans. In some embodimentsthe bacterial host strain is an industrial strain. Numerous bacterialindustrial strains are known and suitable in the present invention. Insome embodiments of the present invention, the bacterial host cell is anAgrobacterium species (e.g., A. radiobacter, A. rhizogenes, and A.rubi). In some embodiments of the present invention, the bacterial hostcell is an Arthrobacter species (e.g., A. aurescens, A. citreus, A.globiformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A.paraffineus, A. protophonniae, A. roseoparqffinus, A. sulfureus, and A.ureafaciens). In some embodiments of the present invention, thebacterial host cell is a Bacillus species (e.g., B. thuringensis, B.anthracis, B. megaterium, B. subtilis, B. lentus, B. circulans, B.pumilus, B. lautus, B.coagulans, B. brevis, B. firmus, B. alkaophius, B.licheniformis, B. clausii, B. stearothermophilus, B. halodurans, and B.amyloliquefaciens). In some embodiments, the host cell is an industrialBacillus strain including but not limited to B. subtilis, B. pumilus, B.licheniformis, B. megaterium, B. clausii, B. stearothermophilus, or B.amyloliquefaciens. In some embodiments, the Bacillus host cells are B.subtilis, B. licheniformis, B. megaterium, B. stearothermophilus, and/orB. amyloliquefaciens. In some embodiments, the bacterial host cell is aClostridium species (e.g., C. acetobutylicum, C. tetani E88, C.lituseburense, C. saccharobutylicum, C. perfringens, and C.beijerinckii). In some embodiments, the bacterial host cell is aCorynebacterium species (e.g., C. glutamicum and C. acetoacidophilum).In some embodiments the bacterial host cell is an Escherichia species(e.g., E. coli). In some embodiments, the host cell is Escherichia coliW3110. In some embodiments, the bacterial host cell is an Erwiniaspecies (e.g., E. uredovora, E. carotovora, E. ananas, E. herbicola, E.punctata, and E. terreus). In some embodiments, the bacterial host cellis a Pantoea species (e.g., P. citrea, and P. agglomerans). In someembodiments the bacterial host cell is a Pseudomonas species (e.g., P.putida, P. aeruginosa, P. mevalonii, and P. sp. D-01 10). In someembodiments, the bacterial host cell is a Streptococcus species (e.g.,S. equisimiles, S. pyogenes, and S. uberis). In some embodiments, thebacterial host cell is a Streptomyces species (e.g., S. ambofaciens, S.achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus,S. fungicidicus, S. griseus, and S. lividans). In some embodiments, thebacterial host cell is a Zymomonas species (e.g., Z. mobilis, and Z.lipolytica).

Many prokaryotic and eukaryotic strains that find use in the presentinvention are readily available to the public from a number of culturecollections such as American Type Culture Collection (ATCC), DeutscheSammlung von Mikroorganismen and Zellkulturen GmbH (DSM), CentraalbureauVoor Schimmelcultures (CBS), and Agricultural Research Service PatentCulture Collection, Northern Regional Research Center (NRRL).

In some embodiments, host cells are genetically modified to havecharacteristics that improve protein secretion, protein stability and/orother properties desirable for expression and/or secretion of a protein.Genetic modification can be achieved by genetic engineering techniquesand/or classical microbiological techniques (e.g., chemical or UVmutagenesis and subsequent selection). Indeed, in some embodiments,combinations of recombinant modification and classical selectiontechniques are used to produce the host cells. Using recombinanttechnology, nucleic acid molecules can be introduced, deleted, inhibitedor modified, in a manner that results in increased yields of uridinephosphorylase variant(s) within the host cell and/or in the culturemedium. For example, knockout of Alpl function results in a cell that isprotease deficient, and knockout of pyr5 function results in a cell witha pyrimidine deficient phenotype. In one genetic engineering approach,homologous recombination is used to induce targeted gene modificationsby specifically targeting a gene in vivo to suppress expression of theencoded protein. In alternative approaches, siRNA, antisense and/orribozyme technology find use in inhibiting gene expression. A variety ofmethods are known in the art for reducing expression of protein incells, including, but not limited to deletion of all or part of the geneencoding the protein and site-specific mutagenesis to disrupt expressionor activity of the gene product. (See e.g., Chaveroche et al., Nucl.Acids Res., 28:22 e97 [2000]; Cho et al., Molec. Plant MicrobeInteract., 19:7-15 [2006]; Maruyama and Kitamoto, Biotechnol Lett.,30:1811-1817 [2008]; Takahashi et al., Mol. Gen. Genom., 272: 344-352[2004]; and You et al., Arch. Microbiol., 191:615-622 [2009], all ofwhich are incorporated by reference herein). Random mutagenesis,followed by screening for desired mutations also finds use (See e.g.,Combier et al., FEMS Microbiol. Lett., 220:141-8 [2003]; and Firon etal., Eukary. Cell 2:247-55 [2003], both of which are incorporated byreference).

Introduction of a vector or DNA construct into a host cell can beaccomplished using any suitable method known in the art, including butnot limited to calcium phosphate transfection, DEAE-dextran mediatedtransfection, PEG-mediated transformation, electroporation, or othercommon techniques known in the art. In some embodiments, the Escherichiacoli expression vector pCK100900i (See, U.S. Pat. No. 9,714,437, whichis hereby incorporated by reference) finds use.

In some embodiments, the engineered host cells (i.e., “recombinant hostcells”) of the present invention are cultured in conventional nutrientmedia modified as appropriate for activating promoters, selectingtransformants, or amplifying the uridine phosphorylase polynucleotide.Culture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and arewell-known to those skilled in the art. As noted, many standardreferences and texts are available for the culture and production ofmany cells, including cells of bacterial, plant, animal (especiallymammalian) and archebacterial origin.

In some embodiments, cells expressing the variant uridine phosphorylasepolypeptides of the invention are grown under batch or continuousfermentations conditions. Classical “batch fermentation” is a closedsystem, wherein the compositions of the medium is set at the beginningof the fermentation and is not subject to artificial alternations duringthe fermentation. A variation of the batch system is a “fed-batchfermentation” which also finds use in the present invention. In thisvariation, the substrate is added in increments as the fermentationprogresses. Fed-batch systems are useful when catabolite repression islikely to inhibit the metabolism of the cells and where it is desirableto have limited amounts of substrate in the medium. Batch and fed-batchfermentations are common and well known in the art. “Continuousfermentation” is an open system where a defined fermentation medium isadded continuously to a bioreactor and an equal amount of conditionedmedium is removed simultaneously for processing. Continuous fermentationgenerally maintains the cultures at a constant high density where cellsare primarily in log phase growth. Continuous fermentation systemsstrive to maintain steady state growth conditions. Methods formodulating nutrients and growth factors for continuous fermentationprocesses as well as techniques for maximizing the rate of productformation are well known in the art of industrial microbiology.

In some embodiments of the present invention, cell-freetranscription/translation systems find use in producing variant uridinephosphorylase(s). Several systems are commercially available and themethods are well-known to those skilled in the art.

The present invention provides methods of making variant uridinephosphorylase polypeptides or biologically active fragments thereof. Insome embodiments, the method comprises: providing a host celltransformed with a polynucleotide encoding an amino acid sequence thatcomprises at least about 70% (or at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about99%) sequence identity to SEQ ID NOs: 2, 4, 246, 594, 776, and/or 868,and comprising at least one mutation as provided herein; culturing thetransformed host cell in a culture medium under conditions in which thehost cell expresses the encoded variant uridine phosphorylasepolypeptide; and optionally recovering or isolating the expressedvariant uridine phosphorylase polypeptide, and/or recovering orisolating the culture medium containing the expressed variant uridinephosphorylase polypeptide. In some embodiments, the methods furtherprovide optionally lysing the transformed host cells after expressingthe encoded uridine phosphorylase polypeptide and optionally recoveringand/or isolating the expressed variant uridine phosphorylase polypeptidefrom the cell lysate. The present invention further provides methods ofmaking a variant uridine phosphorylase polypeptide comprisingcultivating a host cell transformed with a variant uridine phosphorylasepolypeptide under conditions suitable for the production of the varianturidine phosphorylase polypeptide and recovering the variant uridinephosphorylase polypeptide. Typically, recovery or isolation of theuridine phosphorylase polypeptide is from the host cell culture medium,the host cell or both, using protein recovery techniques that are wellknown in the art, including those described herein. In some embodiments,host cells are harvested by centrifugation, disrupted by physical orchemical means, and the resulting crude extract retained for furtherpurification. Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including, but not limited tofreeze-thaw cycling, sonication, mechanical disruption, and/or use ofcell lysing agents, as well as many other suitable methods well known tothose skilled in the art.

Engineered uridine phosphorylase enzymes expressed in a host cell can berecovered from the cells and/or the culture medium using any one or moreof the techniques known in the art for protein purification, including,among others, lysozyme treatment, sonication, filtration, salting-out,ultra-centrifugation, and chromatography. Suitable solutions for lysingand the high efficiency extraction of proteins from bacteria, such as E.coli, are commercially available under the trade name CelLytic B™(Sigma-Aldrich). Thus, in some embodiments, the resulting polypeptide isrecovered/isolated and optionally purified by any of a number of methodsknown in the art. For example, in some embodiments, the polypeptide isisolated from the nutrient medium by conventional procedures including,but not limited to, centrifugation, filtration, extraction,spray-drying, evaporation, chromatography (e.g., ion exchange, affinity,hydrophobic interaction, chromatofocusing, and size exclusion), orprecipitation. In some embodiments, protein refolding steps are used, asdesired, in completing the configuration of the mature protein. Inaddition, in some embodiments, high performance liquid chromatography(HPLC) is employed in the final purification steps. For example, in someembodiments, methods known in the art, find use in the present invention(See e.g., Parry et al., Biochem. J., 353:117 [2001]; and Hong et al.,Appl. Microbiol. Biotechnol., 73:1331 [2007], both of which areincorporated herein by reference). Indeed, any suitable purificationmethods known in the art find use in the present invention.

Chromatographic techniques for isolation of the uridine phosphorylasepolypeptide include, but are not limited to reverse phase chromatographyhigh performance liquid chromatography, ion exchange chromatography, gelelectrophoresis, and affinity chromatography. Conditions for purifying aparticular enzyme will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc.,are known to those skilled in the art.

In some embodiments, affinity techniques find use in isolating theimproved uridine phosphorylase enzymes. For affinity chromatographypurification, any antibody which specifically binds the uridinephosphorylase polypeptide may be used. For the production of antibodies,various host animals, including but not limited to rabbits, mice, rats,etc., may be immunized by injection with the uridine phosphorylase. Theuridine phosphorylase polypeptide may be attached to a suitable carrier,such as BSA, by means of a side chain functional group or linkersattached to a side chain functional group. Various adjuvants may be usedto increase the immunological response, depending on the host species,including but not limited to Freund's (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, dinitrophenol, and potentially useful humanadjuvants such as BCG (Bacillus Calmette Guerin) and Corynebacteriumparvum.

In some embodiments, the uridine phosphorylase variants are prepared andused in the form of cells expressing the enzymes, as crude extracts, oras isolated or purified preparations. In some embodiments, the uridinephosphorylase variants are prepared as lyophilisates, in powder form(e.g., acetone powders), or prepared as enzyme solutions. In someembodiments, the uridine phosphorylase variants are in the form ofsubstantially pure preparations.

In some embodiments, the uridine phosphorylase polypeptides are attachedto any suitable solid substrate. Solid substrates include but are notlimited to a solid phase, surface, and/or membrane. Solid supportsinclude, but are not limited to organic polymers such as polystyrene,polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, andpolyacrylamide, as well as co-polymers and grafts thereof. A solidsupport can also be inorganic, such as glass, silica, controlled poreglass (CPG), reverse phase silica or metal, such as gold or platinum.The configuration of the substrate can be in the form of beads, spheres,particles, granules, a gel, a membrane or a surface. Surfaces can beplanar, substantially planar, or non-planar. Solid supports can beporous or non-porous, and can have swelling or non-swellingcharacteristics. A solid support can be configured in the form of awell, depression, or other container, vessel, feature, or location. Aplurality of supports can be configured on an array at variouslocations, addressable for robotic delivery of reagents, or by detectionmethods and/or instruments.

In some embodiments, immunological methods are used to purify uridinephosphorylase variants. In one approach, antibody raised against awild-type or variant uridine phosphorylase polypeptide (e.g., against apolypeptide comprising any of SEQ ID NOs: 2, 4, 246, 594, 776, and/or868, and/or a variant thereof, and/or an immunogenic fragment thereof)using conventional methods is immobilized on beads, mixed with cellculture media under conditions in which the variant uridinephosphorylase is bound, and precipitated. In a related approach,immunochromatography finds use.

In some embodiments, the variant uridine phosphorylases are expressed asa fusion protein including a non-enzyme portion. In some embodiments,the variant uridine phosphorylase sequence is fused to a purificationfacilitating domain. As used herein, the term “purification facilitatingdomain” refers to a domain that mediates purification of the polypeptideto which it is fused. Suitable purification domains include, but are notlimited to metal chelating peptides, histidine-tryptophan modules thatallow purification on immobilized metals, a sequence which bindsglutathione (e.g., GST), a hemagglutinin (HA) tag (corresponding to anepitope derived from the influenza hemagglutinin protein; See e.g.,Wilson et al., Cell 37:767 [1984]), maltose binding protein sequences,the FLAG epitope utilized in the FLAGS extension/affinity purificationsystem (e.g., the system available from Immunex Corp), and the like. Oneexpression vector contemplated for use in the compositions and methodsdescribed herein provides for expression of a fusion protein comprisinga polypeptide of the invention fused to a polyhistidine region separatedby an enterokinase cleavage site. The histidine residues facilitatepurification on IMIAC (immobilized metal ion affinity chromatography;See e.g., Porath et al., Prot. Exp. Purif., 3:263-281 [1992]) while theenterokinase cleavage site provides a means for separating the varianturidine phosphorylase polypeptide from the fusion protein. pGEX vectors(Promega) may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to ligand-agarose beads (e.g., glutathione-agarose in thecase of GST-fusions) followed by elution in the presence of free ligand.

Accordingly, in another aspect, the present invention provides methodsof producing the engineered enzyme polypeptides, where the methodscomprise culturing a host cell capable of expressing a polynucleotideencoding the engineered enzyme polypeptide under conditions suitable forexpression of the polypeptide. In some embodiments, the methods furthercomprise the steps of isolating and/or purifying the enzymepolypeptides, as described herein.

Appropriate culture media and growth conditions for host cells are wellknown in the art. It is contemplated that any suitable method forintroducing polynucleotides for expression of the enzyme polypeptidesinto cells will find use in the present invention. Suitable techniquesinclude, but are not limited to electroporation, biolistic particlebombardment, liposome mediated transfection, calcium chloridetransfection, and protoplast fusion.

Various features and embodiments of the present invention areillustrated in the following representative examples, which are intendedto be illustrative, and not limiting.

EXPERIMENTAL

The following Examples, including experiments and results achieved, areprovided for illustrative purposes only and are not to be construed aslimiting the present invention. Indeed, there are various suitablesources for many of the reagents and equipment described below. It isnot intended that the present invention be limited to any particularsource for any reagent or equipment item.

In the experimental disclosure below, the following abbreviations apply:M (molar); mM (millimolar), uM and μM (micromolar); nM (nanomolar); mol(moles); gm and g (gram); mg (milligrams); ug and μg (micrograms); L and1 (liter); ml and mL (milliliter); cm (centimeters); mm (millimeters);um and μm (micrometers); sec. (seconds); min(s) (minute(s)); h(s) andhr(s) (hour(s)); U (units); MW (molecular weight); rpm (rotations perminute); psi and PSI (pounds per square inch); ° C. (degreesCentigrade); RT and rt (room temperature); CV (coefficient ofvariability); CAM and cam (chloramphenicol); PMBS (polymyxin B sulfate);IPTG (isopropyl β-D-1-thiogalactopyranoside); LB (lysogeny broth); TB(terrific broth); SFP (shake flask powder); CDS (coding sequence); DNA(deoxyribonucleic acid); RNA (ribonucleic acid); nt (nucleotide;polynucleotide); aa (amino acid; polypeptide); E. coli W3110 (commonlyused laboratory E. coli strain, available from the Coli Genetic StockCenter [CGSC], New Haven, Conn.); HTP (high throughput); HPLC (highpressure liquid chromatography); HPLC-UV (HPLC-Ultraviolet VisibleDetector); 1H NMR (proton nuclear magnetic resonance spectroscopy);FIOPC (fold improvements over positive control); Sigma and Sigma-Aldrich(Sigma-Aldrich, St. Louis, Mo.; Difco (Difco Laboratories, BD DiagnosticSystems, Detroit, Mich.); Microfluidics (Microfluidics, Westwood,Mass.); Life Technologies (Life Technologies, a part of FisherScientific, Waltham, Mass.); Amresco (Amresco, LLC, Solon, Ohio);Carbosynth (Carbosynth, Ltd., Berkshire, UK); Varian (Varian MedicalSystems, Palo Alto, Calif.); Agilent (Agilent Technologies, Inc., SantaClara, Calif.); Infors (Infors USA Inc., Annapolis Junction, Md.); andThermotron (Thermotron, Inc., Holland, Mich.).

EXAMPLE 1 E. coli Expression Hosts Containing Recombinant UridinePhosphorylase Genes

The initial uridine phosphorylase (UP) enzyme used to produce thevariants of the present invention was obtained from the E. coli genomeand cloned into the expression vector pCK110900 (See, FIG. 3 of US Pat.Appln. Publn. No. 2006/0195947), operatively linked to the lac promoterunder control of the lad repressor. The expression vector also containsthe P15a origin of replication and a chloramphenicol resistance gene.The resulting plasmids were transformed into E. coli W3110, usingstandard methods known in the art. The transformants were isolated bysubjecting the cells to chloramphenicol selection, as known in the art(See e.g., U.S. Pat. No. 8,383,346 and WO2010/144103).

EXAMPLE 2 Preparation of HTP UP-Containing Wet Cell Pellets and Lysate

E. coli cells containing recombinant UP-encoding genes from monoclonalcolonies were inoculated into 180 μl ALB containing 1% glucose and 30μg/mL chloramphenicol in the wells of 96-well shallow-well microtiterplates. The plates were sealed with O₂-permeable seals, and cultureswere grown overnight at 30° C., 200 rpm, and 85% humidity. Then, 10 μlof each of the cell cultures were transferred into the wells of 96-welldeep-well plates containing 390 mL TB and 30 μg/mL CAM. The deep-wellplates were sealed with O₂-permeable seals and incubated at 30° C., 250rpm, and 85% humidity until OD₆₀₀ 0.6-0.8 was reached. The cell cultureswere then induced by IPTG to a final concentration of 1 mM and incubatedovernight at 30° C. The cells were then pelleted using centrifugation at4,000 rpm for 10 min. The supernatants were discarded and the pelletsfrozen at −80° C. prior to lysis.

For lysis, 400μ1 lysis buffer containing 100 mM triethanolamine (TEoA)buffer, pH 7.5, 1 g/L lysozyme, and 0.5 g/L polymyxin b sulfate (PMBS)were added to the cell paste in each well. The cells were lysed at roomtemperature for 2 hours with shaking on a bench top shaker. The platewas then centrifuged for 15 min at 4,000 rpm and 4° C. The clearsupernatants were then used in biocatalytic reactions to determine theiractivity levels.

EXAMPLE 3 Preparation of Lyophilized Lysates from Shake Flask (SF)Cultures

Selected HTP cultures grown as described above were plated onto LB agarplates with 1% glucose and 30 μg/ml CAM, and grown overnight at 37° C. Asingle colony from each culture was transferred to 6 ml of LB with 1%glucose and 30 μg/ml CAM. The cultures were grown for 18 hr at 30° C.,250 rpm, and subcultured approximately 1:50 into 250 ml of TB containing30 μg/ml CAM, to a final OD₆₀₀ of 0.05. The cultures were grown forapproximately 195 minutes at 30° C., 250 rpm, to an OD600 between0.6-0.8 and induced with 1 mM IPTG. The cultures were then grown for 20hr at 30° C., 250 rpm. The cultures were centrifuged 4,000 rpm for 20min. The supernatant was discarded, and the pellets were resuspended in30 ml of 20 mM triethanolamine, pH 7.5., and lysed using aMicrofluidizer® processor system (Microfluidics) at 18,000 psi. Thelysates were pelleted (10,000 rpm for 60 min), and the supernatants werefrozen and lyophilized to generate shake flake (SF) enzyme powders.

EXAMPLE 4

Improvements Over SEQ ID NO: 2 in the Phosphorolysis of5′-Isobutyryluridine

SEQ ID NO: 2 was selected as the parent enzyme based on the results ofscreening variants for the phosphorolysis of 5′-isobutyryluridine(Scheme II, below).

The reaction of Scheme II is reversible, and in the initial phase ofwork, the reverse reaction was successfully used as a surrogate for thedesired forward reaction depicted in Scheme I, above.

Libraries of engineered genes were produced using well-establishedtechniques (e.g., saturation mutagenesis, and recombination ofpreviously identified beneficial mutations). The polypeptides encoded byeach gene were produced in HTP as described in Example 2. For allvariants, the cell pellets were lysed by adding 400 μL lysis buffer(containing 100 mM triethanolamine buffer, pH 7.5, 1 g/L lysozyme, and0.5 g/L PMBS) and shaking at room temperature for 2 hours on a tabletopshaker. The plates were centrifuged at 4,000 rpm for 15 minutes at 4° C.to remove cell debris.

Reactions were performed in a 96-well format in 2 mL deep-well plates,with 100 μL, total volume. Reactions included 11.1 g/L (3.5 mM)5′-isobutyryluridine, 10 mM potassium chloride, 50 mM sodium phosphate,pH 7.4, and 10× diluted UP lysate. The reactions were set up as follows:(i) all the reaction components, except for UP, were pre-mixed in asingle solution, and 87.5 μL, of this solution were then aliquoted intoeach well of the 96-well plates (ii) 12.5 μL, of 10x diluted UP lysatewere then added into the wells to initiate the reaction. The reactionplate was heat-sealed with a foil seal and incubated at 35° C. with 600rpm shaking for 18-20 hours. The reactions were quenched with 100 μL ofa 1:1 mixture of acetonitrile in 20 mM TEoA, pH 7.5 buffer. The quenchedreactions were shaken for 3 min on a tabletop shaker followed bycentrifugation at 4,000 rpm for 10 mins at 4° C. to pellet anyprecipitate. Supernatant (50 μL) was then transferred into a 96-wellround bottom plate prefilled with 100 μL of 1:1 mixture of acetonitrilein 20 mM TEoA, pH 7.5 buffer. The samples were analyzed according to theHILIC analytical method summarized in Table 1.1.

TABLE 1.1 HILIC Analytical Method Instrument ThermoScientific U3000 UPLCwith UV Detection Column Xbridge glycan BEH amide, 3.5 μm, 4.6 × 100 mmMobile Phase Isocratic 20:80 water with 0.1% TFA:acetonitrile with 0.1%TFA Flow Rate 1.0 mL/min Run Time 2.25 min Substrate and5′-isobutyryluridine: 0.93 min Product Uracil: 1.25 min Elution orderColumn 25° C. Temperature Injection 2 μL Volume Detection UV 254 nmDetector: Thermo VWD-3400; Peak width 0.02 min; Collection rate = 200Hz; Time Constant = 0.12 s

Activity relative to SEQ ID NO: 2 (Activity FIOP) was calculated as the% conversion to uracil (peak area of uracil/[peak area of uracil+peakarea of 5′-isobutyryluridine]) of the variant compared with the %conversion to uracil produced by the reaction with SEQ ID NO: 2. Theresults are shown in Table 1.2.

TABLE 1.2 FIOP % Uracil Relative SEQ ID to SEQ NO: Amino AcidDifferences ID (nt/aa) (Relative to SEQ ID NO: 2) NO: 2  3/4 E80I +++ 5/6 E80K +++  7/8 V6S +++  9/10 V6G +++  11/12 E80G +++  13/14 H47V +++ 15/16 I147L/H179R/K181Q +++  17/18 I86V/I147L/H179R/K181Q +++  19/20M184R +++  21/22 F7T +++  23/24 K40S/I86V/I147L/K235R/Q236A/H240R +++ 25/26 I228R +++  27/28 K40D/I86V/I147L/K235R/Q236A/H240R +++  29/30N14A/S146E/I147L/K181Q/H240R +++  31/32 Q236A/H240R +++  33/34 E80S +++ 35/36 V6R +++  37/38 K40N/K43F/I86V/I147L/K235R +++  39/40K40S/S146E/I147L/H240R +++  41/42 K40S/I147L/H240R +++  43/44 E80T +++ 45/46 N14A/K40N/I147L/K181Q/M193L/K235R +++  47/48 E80M +++  49/50S146E/I147L/K235R/Q236A +++  51/52 I86V/Q236A/H240R +++  53/54 H47V/T88A+++  55/56 I147L/K235R/H240R/A249V +++  57/58 N14A/S146E/Q236A/H240R ++ 59/60 I86V/C136V/S146E/I147L/H179R/K181Q ++  61/62K43F/I147L/H179R/K181Q ++  63/64 K405/Q236A/H240R ++  65/66 H179R/K181Q++  67/68 K40D/I86V/K235R/Q236A/H240R ++  69/70K40N/I86V/H179R/K235R/H240R ++  71/72 I228K ++  73/74 S146E/Q236A/H240R++  75/76 S73G ++  77/78 A97T ++  79/80 V6Y ++  81/82I86V/K235R/Q236A/H240R ++  83/84 V6W ++  85/86N14A/H179R/K181Q/M193L/K235R/H240R ++  87/88 H179R/K181Q/M193L/H240R ++ 89/90 K181Q ++  91/92 I86V/H240R ++  93/94 K40S/K235R/Q236A/H240R ++ 95/96 N14A/I147L/M193L/K235R/Q236A/H240R ++  97/98N14A/K40S/C136V/H179R/Q236A/H240R ++  99/100K40N/K43F/I147L/H179R/Q236A/H240R ++ 101/102 L9M ++ 103/104I86V/C136V/S146E/I147L/H179R/K235R/Q236A ++ 105/106 S146E/I147L ++107/108 M38L/K40S/I86V/I147L/Q236A/H240R ++ 109/110N14A/M38L/I86V/S146E/I147L/K235R/Q236A/H240R ++ 111/112 A231I ++ 113/114M38L/K40D/I86V/S146E/I147L/H179R/K181Q ++ 115/116K40S/K43F/I147L/Q236A/H240R ++ 117/118 S146E/H240R ++ 119/120 H240R ++121/122 N14A/K235R/Q236A ++ 123/124N14A/K40D/I86V/I147M/M193L/Q236A/H240R ++ 125/126 A231W ++ 127/128 H47R++ 129/130 V64C ++ 131/132 S146E/K235R/H240R ++ 133/134 T237V ++ 135/136H47M ++ 137/138 V31T + 139/140 V421/K235R/Q236A + 141/142 M184S +143/144 K40S/K43F/I147L/H179R/H240R + 145/146 V216M + 147/148N14A/K40D/K235R + 149/150 I92V + 151/152 K235R/Q236A + 153/154K43F/I86V/I147L/K181Q/H240R + 155/156 N103G/A249V + 157/158 I228L +159/160 V6F + 161/162 N14A/K40S + 163/164 N14A/K40N + 165/166 S239A +167/168 I228A + 169/170 N14A + 171/172 K40D/K235R + 173/174 A231P +175/176 K43F/S146E/I147L/K235R/Q236A/H240R + 177/178 T110A + 179/180N14A/K40N/S146E/K235R/Q236A + 181/182 K43F/I147L + 183/184 K40D +185/186 A231Y + 187/188 T110C + 189/190 K40S/Q236A + 191/192I86V/K235R + 193/194 Q236A + 195/196N14A/M38L/K40S/S146E/I147L/H179R/K235R/Q236A + 197/198 S239K + 199/200K40S/I86V/S146E/K235R/Q236A + 201/202 I228G + 203/204 V104T + 205/206K235R + 207/208 D106E + 209/210 K40N/K43F/S146E/I147L + 211/212K40N/K43F/I86V/S146E/H240R = 213/214 D29S + 215/216 V245S + 217/218Q226R + 219/220 N14A/I86V/S146E + 221/222 G105M + 223/224 H179R +225/226 A231T + 227/228 T233S + 229/230 S239R + 231/232 Q99E + 233/234A157S + 235/236 T110S + 237/238 I86V + 239/240 E167S + 241/242K43F/S146E/H179R/H240R + 243/244 N103S + Levels of increased activitywere determined relative to the reference polypeptide of SEQ ID NO: 2and defined as follows: “+” 1.00 to 1.05, “++” > 1.05, “+++” > 1.15

EXAMPLE 5 Improvements Over SEQ ID NO: 4 toward the synthesis of5′-isobutyryluridine

SEQ ID NO: 4 was selected as the parent enzyme based on the results ofscreening variants toward the synthesis of 5′-isobutyryluridine from5′-isobutyryl ribose-1-phosphate and uracil (Scheme I, above).

Libraries of engineered genes were produced using well-establishedtechniques (e.g., saturation mutagenesis, and recombination ofpreviously identified beneficial mutations). The polypeptides encoded byeach gene were produced in HTP as described in Example 2. For allvariants, the cell pellets were lysed by adding 400 μL lysis buffer(containing 100 mM triethanolamine buffer, pH 7.5, 1 g/L lysozyme, and0.5 g/L PMBS) and shaking at room temperature for 2 hours on a tabletopshaker. The plates were centrifuged at 4,000 rpm for 15 minutes at 4° C.to remove cell debris.

Reactions were performed in a 96-well format in 2 mL deep-well plates,with 100 μL, total volume. Reactions included 67 mM 5′-isobutyrylribose-1-phosphate solution, 7.5 g/L (67 mM) uracil, 91 g/L (267 mM, 4equiv.) sucrose, 0.019 g/L (0.25 wt % wrt uracil) SUP-101 (SEQ ID NO:852), 50 mM triethanolamine, pH 7.5, and 100× diluted UP lysate. Thereactions were set up as follows: (i) all the reaction components,except for UP, were pre-mixed in a single solution, and 90 μL, of thissolution were then aliquoted into each well of the 96-well plates (ii)10 μL, of 100× diluted UP lysate were then added into the wells toinitiate the reaction. The reaction plate was heat-sealed with a foilseal and incubated at 30° C. with 600 rpm shaking for 18-20 hours. Thereactions were quenched with 400 μL, of a 1:1 mixture of acetonitrile in20 mM TEoA, pH 7.5 buffer. The quenched reactions were shaken for 5 minon a tabletop shaker followed by centrifugation at 4,000 rpm for 10 minsat 4° C. to pellet any precipitate. Supernatant (30 μL) was thentransferred into a 96-well round bottom plate prefilled with 120 μL, of1:9 mixture of acetonitrile in 20 mM TEoA, pH 7.5 buffer. The sampleswere analyzed according to the reverse-phase analytical methodsummarized in Table 2.1.

TABLE 2.1 Reverse-Phase Analytical Method Instrument ThermoFisherVanquish UPLC with UV Detection Column Atlantis HSS T3, 1.8 μm, 2.1 ×100 mm Mobile Phase Mobile Phase A: water with 0.1% TFA Mobile Phase B:acetonitrile with 0.1% TFA Gradient 0-1.0 min 5% B, 1.0-1.1 min 50% B,1.1-2.7 50% B, 2.7-2.8 min 5% B, 2.8-3.25 min 5% B Flow Rate 0.3 mL/minRun Time 3.25 min Substrate and Product Uracil: 1.00 min Elution order5′-isobutyryluridine: 2.52 min Column Temperature 40° C. InjectionVolume 10 μL Detection UV 254 nm Detector: Vanquish Diode Array DetectorHL; Peak width 0.2 min; Collection rate = 2 Hz; Time Constant = 2 s

Activity relative to SEQ ID NO: 4 (Activity FIOP) was calculated as thepeak area of 5′-isobutyryluridine product formed by the reaction withthe variant compared with the peak area of 5′-isobutyryluridine productproduced by the reaction with SEQ ID NO: 4. The results are shown inTable 2.2.

TABLE 2.2 FIOP 5-IBU SEQ ID Relative NO: Amino Acid Differences to SEQID (nt/aa) (Relative to SEQ ID NO: 4) NO: 4 245/246 A241M +++ 247/248K3E/V31T/H47V/H179R/K181Q/V216M +++ 249/250 K3E/H47V/H179R/K181Q/V216M+++ 251/252 H47W +++ 253/254 K3E/V31T/H47V/H179R/V216M +++ 255/256H47V/H179R/K181Q/V216M +++ 257/258 D160G +++ 259/260K3E/V31T/H47V/H179R/V216M/T237V +++ 261/262 H47V/H179R/V216M +++ 263/264H179R/K181Q/V216M/T237V +++ 265/266 V31T/H47V/H179R/V216M +++ 267/268K3E/H47V/H179R/V216M/T237V +++ 269/270 H47V/K181Q/V216M/T237V +++271/272 E238P +++ 273/274 E185A +++ 275/276K3E/V31T/H47V/H179R/K181Q/T237V +++ 277/278 H179R/K181Q/V216M +++279/280 T233S +++ 281/282 L108V +++ 283/284 H47V/K181Q/V216M +++ 285/286V31T/H179R/V216M +++ 287/288 S183L +++ 289/290 V31T/K181Q/V216M/T237V+++ 291/292 G218V +++ 293/294 V31T/H47V/H179R/K181Q/T237V +++ 295/296K3E/H179R/V216M/T237V +++ 297/298 K3E/H179R/K181Q/V216M +++ 299/300H179R/V216M +++ 301/302 K3E/H179R/V216M +++ 303/304 K181Q/V216M/T237V+++ 305/306 H47V/H179R/K181Q +++ 307/308 H47V +++ 309/310 V216M +++311/312 V31T/H47V/V216M +++ 313/314 H47V/V216M +++ 315/316 P100G +++317/318 K3E/V31T/H179R/V216M +++ 319/320 T95S +++ 321/322 L108A +++323/324 V31T/V216M +++ 325/326 I228L ++ 327/328 V31T/K181Q/V216M ++329/330 V216M/T237V ++ 331/332 T111K ++ 333/334 K3E/H179R/K181Q/T237V ++335/336 H240G ++ 337/338 K3E/V31T/H179R/K181Q/T237V ++ 339/340R87H/D160C ++ 341/342 I92M ++ 343/344 T233G ++ 345/346 V31T/H47V/K181Q++ 347/348 A241L ++ 349/350 D106E ++ 351/352 P100E ++ 353/354 K235S ++355/356 Q188A ++ 357/358 G191F ++ 359/360 K181Q/V216M ++ 361/362 K33Y ++363/364 H179R/T237V ++ 365/366 V24L ++ 367/368 V155R ++ 369/370 K33V ++371/372 E238S ++ 373/374 T233N ++ 375/376 H101A ++ 377/378K3E/V31T/K181Q/V216M ++ 379/380 T151S ++ 381/382 Q236A/T237V/H240R ++383/384 P100D ++ 385/386 H152L ++ 387/388 K3E/K181Q/V216M/T237V ++389/390 V31T/V216M/T237V ++ 391/392 H101T ++ 393/394 P100T ++ 395/396A189L ++ 397/398 T111R ++ 399/400 G191R ++ 401/402 K33R ++ 403/404 Q236P++ 405/406 T67G ++ 407/408 V177R ++ 409/410 G218A ++ 411/412 H179R/K181Q++ 413/414 V24M ++ 415/416 V31T/H179R/K181Q ++ 417/418 A241G ++ 419/420K3E ++ 421/422 S159T ++ 423/424 E185W ++ 425/426 H179R/K181Q/A241V ++427/428 E238G ++ 429/430 H101W ++ 431/432 H179R ++ 433/434 E185R ++435/436 V31T/K181Q/T237V ++ 437/438 T201L ++ 439/440 I228T ++ 441/442S46Q ++ 443/444 K3E/L9M/V216M ++ 445/446 V31T/Q236A/T237V/H240R ++447/448 P100R ++ 449/450 K3E/V31T/H179R/K181Q ++ 451/452 H101V ++453/454 K3E/H179R/K181Q ++ 455/456 E185V ++ 457/458 E185G ++ 459/460A189V ++ 461/462 K3E/K181Q/V216M ++ 463/464 Q236T ++ 465/466K3E/H47V/K181Q ++ 467/468 A241P ++ 469/470 K3E/V216M/Q236A/H240R ++471/472 V31T/T237V ++ 473/474 S159A ++ 475/476 A189R ++ 477/478K181Q/T237V ++ 479/480 A231V ++ 481/482 H47V/I147L/K181Q/A231I ++483/484 K3E/L9M/K235R/T237V ++ 485/486 E185H ++ 487/488 L108M + 489/490H47V/H179R/M184R + 491/492 V31T + 493/494 K235H + 495/496 E185Q +497/498 A97S + 499/500 K3E/H179R/T237V + 501/502 K3E/V31T/H179R +503/504 K3E/L9M/V216M/Q236A + 505/506 H47V/K181Q/A231W + 507/508 E185K +509/510 V31T/H179R + 511/512 Q99V + 513/514 Q236S + 515/516 T137G +517/518 S183W + 519/520 N222L + 521/522 T52W + 523/524 Q236A + 525/526T233A + 527/528 V31T/H47V + 529/530 I63M + 531/532 T237V + 533/534E185S + 535/536 Q99L + 537/538 V31T/K181Q + 539/540 K33L + 541/542A241W + 543/544 S173R + 545/546 Q188L + 547/548 I228H + 549/550 V24Q +551/552 H152S + 553/554 G105S + 555/556 K33N + 557/558 T52L + 559/560Q83G + 561/562 N222R + 563/564 A248T + 565/566 K3E/H179R + 567/568K235R/T237V + 569/570 S183T + 571/572 H152V + 573/574 Q99R + 575/576D170A + 577/578 K13L + 579/580 A231G + 581/582 H179R/I228A/A231I +583/584 H179R/K181Q/A231W + 585/586 L9M/V216M/Q236A/T237V + 587/588K3E/V31T/K181Q/T237V + 589/590 L9M/T237V + 591/592 K181Q + Levels ofincreased activity were determined relative to the reference polypeptideof SEQ ID NO: 4 and defined as follows: “+” 1.06 to 1.15, “++” > 1.15,“+++” > 1.3

EXAMPLE 6 Improvements Over SEQ ID NO: 246 Toward the Synthesis of5′-Isobutyryluridine

SEQ ID NO: 246 was selected as the parent enzyme based on the results ofscreening variants toward the synthesis of 5′-isobutyryluridine(compound (1)) from 5′-isobutyryl ribose-1-phosphate (compound (2)) anduracil (compound (3))(Scheme I).

Libraries of engineered genes were produced using well-establishedtechniques (e.g., saturation mutagenesis, and recombination ofpreviously identified beneficial mutations). The polypeptides encoded byeach gene were produced in HTP as described in Example 2. For allvariants, the cell pellets were lysed by adding 400 μL, lysis buffer(containing 100 mM triethanolamine buffer, pH 7.5, 1 g/L lysozyme, and0.5 g/L PMBS) and shaking at room temperature for 2 hours on a tabletopshaker. The plates were centrifuged at 4,000 rpm for 15 minutes at 4° C.to remove cell debris.

Reactions were performed in a 96-well format in 2 mL deep-well plates,with 100 μL total volume. Reactions included 67 mM 5′-isobutyrylribose-1-phosphate solution, 7.5 g/L (67 mM) uracil, 91 g/L (267 mM, 4equiv.) sucrose, 0.019 g/L (0.25 wt % wrt uracil) SUP-101 (SEQ IDNO:852), 50 mM triethanolamine, pH 7.5, and 100× diluted UP lysate. Thereactions were set up as follows: (i) all the reaction components,except for UP, were pre-mixed in a single solution, and 90 μL, of thissolution were then aliquoted into each well of the 96-well plates (ii)10 μL, of 100× diluted UP lysate were then added into the wells toinitiate the reaction. The reaction plate was heat-sealed with a foilseal and incubated at 30° C. with 600 rpm shaking for 18-20 hours. Thereactions were quenched with 400 μL of a 1:1 mixture of acetonitrile in20 mM TEoA, pH 7.5 buffer. The quenched reactions were shaken for 5 minon a tabletop shaker followed by centrifugation at 4,000 rpm for 10 minsat 4° C. to pellet any precipitate. Supernatant (30 μL) was thentransferred into a 96-well round bottom plate prefilled with 120 μL of1:9 mixture of acetonitrile in 20 mM TEoA, pH 7.5 buffer. The sampleswere analyzed according to the reverse-phase analytical methodsummarized in Table 2.1.

Activity relative to SEQ ID NO: 246 (Activity FIOP) was calculated asthe peak area of 5′-isobutyryluridine product formed by the reactionwith the variant compared with the peak area of 5′-isobutyryluridineproduct produced by the reaction with SEQ ID NO: 246. The results areshown in Table 3.1.

TABLE 3.1 FIOP 5-IBU SEQ ID Relative NO: Amino Acid Differences to SEQID (nt/aa) (Relative to SEQ ID NO: 246) NO: 246 593/594K3E/V24L/H47W/A189R/I228L/T233S +++ 595/596 K3E/H47V/P100E/D160G/E185A+++ 597/598 K3E/H47W/S183L/A189R/I228L/T233S +++ 599/600K3E/V24L/H47W/P100E +++ 601/602 K3E/V24L/H47W/L108V/D160G/M241A +++603/604 P100E/D160G/E185A +++ 605/606 K3E/H47W/P100E/E185A/A189R/I228L+++ 607/608 V24L/P100E/D160G/T233S +++ 609/610 K3E/H47V/T95S/D160G/A189R+++ 611/612 H47W/P100E/A189R/T233S +++ 613/614 K3E/V24L/H47W/D160G/A189R+++ 615/616 K3E/D160G +++ 617/618 K3E/V24L/S183L/E185A/V216M +++ 619/620K3E/V24L/K33Y/H47V/P100E/V216M/I228L/T233S +++ 621/622K3E/H47W/T95S/P100E/L108V/A189R/T233S +++ 623/624K3E/K33Y/H47W/P100E/L108V/A189R/V216M/I228L/T233S +++ 625/626V24L/H47V/V216M ++ 627/628 K3E/V24L/H47V/I228L/T233S ++ 629/630L108V/E185A/V216M/I228L/T233S ++ 631/632 K3E/H47W/A189R ++ 633/634K3E/V24L/H47V/I228L ++ 635/636 K3E/K33Y/H47V/P100E/V216M ++ 637/638K3E/V24L/K33Y/H47W/P100E/S183L/E185A ++ 639/640 V24L/H47V/T95S/P100E ++641/642 K3E/H47W/L108V/T111K ++ 643/644 H47V/L108V/D160G/I228L/M241A ++645/646 K3E/H47W/I228L/T233S ++ 647/648 H47V/P100E/A189R ++ 649/650K3E/V24L/P100E ++ 651/652 K3E/H47W ++ 653/654 K3E/V24L/T111K/I228L/T233S++ 655/656 K3E/V24L/H47W/P100E/L108V/T111K/D160G/E185A/T233S/M241A ++657/658 K3E/V24L/P100E/D160G/G218V/M241A ++ 659/660H47V/P100E/T111K/T233S ++ 661/662 K3E/V24L/A189R/T233S ++ 663/664H47V/D160G/E185A/A189R/T233S ++ 665/666 K3E/T95S/P100E/D160G/I228L/T233S++ 667/668 H47V/T111K ++ 669/670K3E/H47W/P100E/L108V/S183L/E185A/A189R/M241A ++ 671/672K3E/K33Y/H47W/L108V/A189R/T233S/M241A ++ 673/674 V24L/T95S/S183L ++675/676 K3E/S183L/E185A/A189R/I228L/T233S ++ 677/678 T95S/P100E/S183L ++679/680 K3E/V24L/T111K ++ 681/682 H47V/I228L/T233S ++ 683/684 L108V ++685/686 V24L/H47V/T95S/P100E/D160G/I228L/T233S/M241A ++ 687/688V24L/A189R/I228L/T233S ++ 689/690 P100E/I228L/T233S ++ 691/692L108V/S183L/A189R/T233S ++ 693/694 V24L ++ 695/696 T95S/P100E/T233S ++697/698 V24L/K33Y/H47W/I228L/M241A ++ 699/700 K3E/V24L/T95S/P100E ++701/702 K3E/T95S/T233S ++ 703/704 V24L/K33Y/H47W + 705/706 K3E/A189R +707/708 K33Y/H47W/T95S/P100E/T233S/M241A + 709/710 K3E/E185A/A189R +711/712 K3E/H47V/T95S/P100E/T111K/M241A + 713/714K3E/V24L/K33Y/P100E/S183L/E185A/I228L + 715/716K3E/T95S/P100E/S183L/V216M/I228L/T233S + 717/718K33Y/P100E/S183L/E185A + 719/720 H47V + 721/722K3E/K33Y/H47W/T95S/P100E/M241A + 723/724 K3E/S183L/A189R/I228L/T233S +725/726 V24L/K33Y/P100E/L108V/M241A + 727/728 T95S/P100E/I228L/T233S +729/730 D160G/T233S + 731/732 K3E/T95S/E185A/A189R/V216M + 733/734I228L/T233S + 735/736 I228L + 737/738 K3E/T95S/P100E/S183L/T233S +739/740 K3E/K33Y/D160G/T233S + 741/742 K33Y/H47W/T233S + 743/744K3E/K33Y/H47V/P100E/T111K/T233S/M241A + 745/746K3E/V24L/T111K/S183L/I228L/T233S/M241A + 747/748K33Y/H47V/T95S/P100E/T233S + 749/750K3E/V24L/T95S/P100E/D160G/A189R/I228L/M241A + 751/752 K3E/E185A +753/754 T95S/P100E/I228L + 755/756 V24L/H47V/E185A/V216M/G218V + 757/758K3E/T95S/A189R + 759/760 K33Y/P100E/E185A/T2335 + 761/762K3E/V24L/K33Y/L108V + 763/764 K3E/K33Y/H47V/P100E/T111K/I228L + 765/766K3E/K33Y/H47W/L108V/T111K/T2335 + 767/768 V24L/D160G/5183L/E185A +769/770 K33Y/H47W/D160G + 771/772 T955/P100E/A189R + Levels of increasedactivity were determined relative to the reference polypeptide of SEQ IDNO: 246 and defined as follows: “+” 1.05 to 1.2, “++” > 1.2, “+++” > 1.6

EXAMPLE 7 Improvements Over SEQ ID NO: 594 Toward the Synthesis of5′-Isobutyryluridine

SEQ ID NO: 594 was selected as the parent enzyme based on the results ofscreening variants toward the synthesis of 5′-isobutyryluridine(compound (1)) from 5′isobutyryl ribose-1-phosphate (compound (2)) anduracil (compound (3))(Scheme I).

Libraries of engineered genes were produced using well-establishedtechniques (e.g., saturation mutagenesis, and recombination ofpreviously identified beneficial mutations). The polypeptides encoded byeach gene were produced in HTP as described in Example 2. For allvariants, the cell pellets were lysed by adding 400 μL, lysis buffer(containing 100 mM triethanolamine buffer, pH 7.5, 1 g/L lysozyme, and0.5 g/L PMBS) and shaking at room temperature for 2 hours on a tabletopshaker. The plates were centrifuged at 4,000 rpm for 15 minutes at 4° C.to remove cell debris.

Reactions were performed in a 96-well format in 2 mL deep-well plates,with 100 μL total volume. Reactions included 67 mM 5′-isobutyrylribose-1-phosphate solution, 7.5 g/L (67 mM) uracil, 91 g/L (267 mM, 4equiv.) sucrose, 0.019 g/L (0.25 wt % wrt uracil) SUP-101 (SEQ IDNO:852), 50 mM triethanolamine, pH 7.5, and 100× diluted UP lysate. Thereactions were set up as follows: (i) all the reaction components,except for UP, were pre-mixed in a single solution, and 90 μL of thissolution were then aliquoted into each well of the 96-well plates (ii)10 μL, of 100× diluted UP lysate were then added into the wells toinitiate the reaction. The reaction plate was heat-sealed with a foilseal and incubated at 30° C. with 600 rpm shaking for 18-20 hours. Thereactions were quenched with 400 μL of a 1:1 mixture of acetonitrile in20 mM TEoA, pH 7.5 buffer. The quenched reactions were shaken for 5 minon a tabletop shaker followed by centrifugation at 4,000 rpm for 10 minsat 4° C. to pellet any precipitate. Supernatant (30 μL) was thentransferred into a 96-well round bottom plate prefilled with 120 μL of1:9 mixture of acetonitrile in 20 mM TEoA, pH 7.5 buffer. The sampleswere analyzed according to the reverse-phase analytical methodsummarized in Table 2.1.

Activity relative to SEQ ID NO: 594 (Activity FIOP) was calculated asthe peak area of 5′-isobutyryluridine product formed by the reactionwith the variant compared with the peak area of 5′-isobutyryluridineproduct produced by the reaction with SEQ ID NO: 594. The results areshown in Table 4.1.

TABLE 4.1 FIOP 5-IBU SEQ Relative ID NO: Amino Acid Differences to SEQID (nt/aa) (Relative to SEQ ID NO: 594) NO: 594 773/774V6S/S46Q/T52S/I63V/A97T/L121G/L126M/H179R +++ 775/776V6S/L9M/D29A/K40N/P100G/L121G/L126M/H179R/ +++ K181Q/R189E/T237V 777/778V6S/L9M/L121G/H179R/K181Q +++ 779/780 V6S/T52S/F180L/K181Q +++ 781/782L9M/A97T/P100G/D106E/L126M/F180L/A207S/A231E +++ 783/784L9M/K40N/S46Q/A97T/P100G/D106E/E135D/H179R/ +++ K181Q/A207S/A231E785/786 V6S/163V/L126M/H179R/V242I +++ 787/788 T52S/H179R/R189E +++789/790 L126M/F180L/R189E +++ 791/792 T52S/I63V/L126M ++ 793/794 V6S ++795/796 H179R ++ 797/798 F180L ++ 799/800 L9M ++ 801/802 A207S ++803/804 L9M/T52S/L126M/R189E/V242I ++ 805/806 T237V ++ 807/808 I63V ++809/810 R189E ++ 811/812 A97T ++ 813/814 T52S ++ 815/816 L126M ++817/818 K181Q ++ 819/820 L121G ++ 821/822 S46Q ++ 823/824 A231E ++825/826 E135D ++ 827/828 T201L + 829/830 R189D + 831/832 V242I + 833/834D29A + 835/836 P100G + 837/838 L9M/K181Q/V242I + 839/840 K40N + 841/842D106E + 843/844 E142A + 845/846 P61A + 847/848 Q236E + 849/850 N230D +Levels of increased activity were determined relative to the referencepolypeptide of SEQ ID NO: 594 and defined as follows: “+” 1.02 to 1.1,“++” > 1.1, “+++” > 1.5

EXAMPLE 8 Improvements Over SEQ ID NO: 776 Toward the Synthesis of5′-Isobutyryluridine

SEQ ID NO: 776 was selected as the parent enzyme based on the results ofscreening variants toward the synthesis of 5′-isobutyryluridine(compound (1)) from 5′-isobutyryl ribose (compound (4)) and uracil(compound (3))(Scheme III).

The uridine phosphorylase reaction is reversible. Initial experimentslooked at the “reverse” reaction or the phosphorolysis of compound (1)via the UP enzyme reaction (Scheme II). Subsequent experimentsinvestigated the “forward” reaction or the generation of compound (1)from compounds (2) and (3) (as shown in Scheme I). In addition, UPenzyme variants were also investigated in the “forward” direction inreactions with in situ generation of compound (2). In this example,compound (2) is generated in reaction via an evolved 5-Methylthioribose(MTR) kinase which catalyzes the phosphorylation of the C-1 hydroxylgroup of the ribose moiety of compound (4). The UP enzyme variants inthe same reaction can be analyzed for their ability to convert compound(2) generated from MTR kinase to the form compound (1). The overallcascade reaction is summarized in Scheme III, above. The cascadereaction described below also includes auxiliary enzymes to support thekinase reaction: (1) pyruvate oxidase (with TPP, FAD, and pyruvate) bothconsumes phosphate (driving the equilibrium toward products) andgenerates acetyl-phosphate, (2) catalase consumes peroxide by-productformed by pyruvate oxidase, and (3) acetate kinase produces ATP (fromADP+acetyl-phosphate) which is subsequently used by the evolved MTRkinase. UP enzyme variants were screened in this cascade since it isexemplary of a possible scaled process. Other similar enzyme cascadesare also viable.

Libraries of engineered genes were produced using well-establishedtechniques (e.g., saturation mutagenesis, and recombination ofpreviously identified beneficial mutations). The polypeptides encoded byeach gene were produced in HTP as described in Example 2. For allvariants, the cell pellets were lysed by adding 400 μL, lysis buffer(containing 100 mM triethanolamine buffer, pH 7.5, 1 g/L lysozyme, and0.5 g/L PMBS) and shaking at room temperature for 2 hours on a tabletopshaker. The plates were centrifuged at 4,000 rpm for 15 minutes at 4° C.to remove cell debris.

Reactions were performed in a 96-well format in 2 mL deep-well plates,with 100 μL total volume. Reactions included 72.6 g/L (330 mM)5′-isobutyryl ribose solution (19.5 wt %), 37 g/L (330 mM) uracil, 0.76g/L (0.5 mol%) TPP, 0.94 g/L (0.5 mol %) ATP, 0.14g/L (0.05 mol%) FAD,46.3 g/L (412 mM) pyruvic acid, 3.7 g/L (33 mM) K2HPO4, 0.5 wt % acetatekinase (ACK-101 Codexis, Inc.), 1.9 wt% evolved MTR-kinase (SEQ ID NO:1198), 0.5 wt % catalase, 0.8 wt % pyruvate oxidase (SEQ ID NO: 1200),and 10 mM MgCl₂. The reactions were set up as follows: (i) an aqueoussolution of pyruvic acid and monopotassium phosphate was cooled to 0° C.before addition of 5′-isobutyryl ribose-1-phosphate solution(portion-wise). The resultant solution was pH adjusted to pH 7.2 using8N KOH while maintained at 0° C. To this solution was added: MgC1₂, TPP,FAD, and ATP followed by AcK, MTR-kinase, catalase, and pyruvateoxidase. The resulting solution (90 μL) was then aliquoted into eachwell of the 96-well plates. (ii) 10 μL of 100x diluted UP lysate wasthen added into the wells to initiate the reaction. The reaction platewas sealed with air-permeable tape and incubated at 25 ° C. with 250 rpmshaking for 18-20 hat 85% humidity. The reactions were quenched with1000 μL of a 1:1 mixture of acetonitrile in 20 mM TEoA, pH 7.5 buffer.The quenched reactions were shaken vigorously for 15 min on a tabletopshaker followed by centrifugation at 4,000 rpm for 10 mins at roomtemperature to pellet any precipitate. Supernatant (15 μL) was thentransferred into a 96-well round bottom plate prefilled with 120 μL, of1:9 mixture of acetonitrile in 20 mM TEoA, pH 7.5 buffer. The sampleswere analyzed according to the reverse-phase analytical methodsummarized in Table 5.1 below.

TABLE 5.1 Reverse-Phase Analytical Method Instrument ThermoScientificU3000 UPLC with UV Detection Column Atlantis HSS T3, 1.8 μm, 2.1 × 100mm Mobile Phase Mobile Phase A: water with 0.1% TFA Mobile Phase B:acetonitrile with 0.1% TFA Gradient 0-1.0 min 5% B, 1.0-1.1 min 50% B,1.1-2.7 50% B, 2.7-2.8 min 5% B, 2.8-3.25 min 5% B Flow Rate 0.3 mL/minRun Time 3.25 min Substrate and Uracil: 1.00 min Product Elution5′-isobutroyl uridine: 2.52 min order Column 40° C. TemperatureInjection 2 μL Volume Detection UV 254 nm Detector: Thermo VWD-3400;Peak width 0.02 min; Collection rate = 200 Hz; Time Constant = 0.12 s

Activity relative to SEQ ID NO: 776 (Activity FIOP) was calculated asthe peak area of 5′-isobutyryluridine product formed by the reactionwith the variant compared with the peak area of 5′-isobutyryluridineproduct produced by the reaction with SEQ ID NO: 776. The results areshown in Table 5.2.

TABLE 5.2 FIOP 5-IBU SEQ ID Relative NO: Amino Acid Differences to SEQID (nt/aa) (Relative to SEQ ID NO: 776) NO: 776  851/852A45G/T51I/E189V/S233D +++  853/854 S6T/A45G/T51I/S208V/S233D +++ 855/856 A45G/T51I/A144G/S208V/Q226G/A231V/S233D +++  857/858T51I/M126Y/A144G/Q226G/A231V/S233D/Q236W +++  859/860A45G/T51I/S208V/S233D +++  861/862 A45G/Q188R/E189Y/A231V/S233D +++ 863/864 S6T/T51I/M126Y/E189Y/S208V/S233D/Q236G +++  865/866S6T/M126Y/Q188R/A231V/S233D +++  867/868 A45G/T51I/Q188R/E189V +++ 869/870 S6T/T51I/M126Y/E189V/S233D +++  871/872S6T/A45G/T51I/Q188R/E189Y +++  873/874 S6T/A45G/T51I/E189Y/S208V +++ 875/876 S6T/A45G/T51I/A144G +++  877/878A45G/T51I/M126Y/A144G/S208V/Q226G/L228M +++  879/880S6T/T51I/S208V/A231V +++  881/882 S6T/T51I/S208V/Q226G/S233D +++ 883/884 S6T/A45G/T51I/E189V/L228M/Q236W +++  885/886A45G/A144G/Q226G/A231V/S233D +++  887/888 S6T/T51I/Q188R/E189Y/Q236G +++ 889/890 T51I/S233D +++  891/892 A45G/Q188R/E189Y/Q226G/L228M +++ 893/894 A45G/T51I/S208V/Q226G/A231V +++  895/896 A45G/Q188R/E189Y +++ 897/898 A45G/T51I/A144G +++  899/900 S208V/A231V/S233D +++  901/902A45G/Q188R/E189Y/S208V/L228M ++  903/904 A45G/M126Y/E189Y ++  905/906A45G/T51I ++  907/908 T51I/A144G ++  909/910 T51I/E189V/S208V/Q226G ++ 911/912 H8A/E142L/I147C ++  913/914 T51I/Q188R/E189Y/L228M ++  915/916A45G/M126Y/E189Y/S208V/Q226G ++  917/918S6T/A45G/A149T/Q188R/E189Y/S208V/S233D/Q236G ++  919/920A45G/E189V/S208V ++  921/922 S6T/T51I/E189Y/S208V/A231V/S233D ++ 923/924 A144G/Q188R/L228M ++  925/926 S208V/L228M ++  927/928S6T/T51I/Q188R/E189Y/S208V/Q226G/L228M ++  929/930 A45G/E189V ++ 931/932 S233D ++  933/934 T51I/S208V ++  935/936 T51I/M126Y/S208V ++ 937/938 A36I/A143G/I147C/Q181R ++  939/940 H8A/A36I/A143G/I147M/K235G++  941/942 K235P ++  943/944 A143G/I147C/K235G ++  945/946S208V/Q226G/S233D ++  947/948 H8A/I147M/K235G ++  949/950S6T/Q188R/E189Y/S208V/L228M/S233D ++  951/952 T51I/M126Y/A144G/S208V ++ 953/954 S6T/A144G/S208V ++  955/956 S6T/T51I/M126Y/E189Y/A231V/Q236W ++ 957/958 A45G/E189Y ++  959/960 A45G/A144G/E189V/L228M ++  961/962A36I/A143G/I147C/K235G ++  963/964 T51I/A144G/Q226G ++  965/966S6T/A45G/T51I/L81W/M126Y/Q226G/S233D ++  967/968 H8A/A36I/Q181R/K235G ++ 969/970 A45G ++  971/972 S6T/T51I/M126Y/A144G/S208V ++  973/974Q188R/E189V/S233D ++  975/976 A22G/I147C/Q181R/K235G/R250A ++  977/978E3N/H8A/A22G/A36I/Q181R/K235G/R250A ++  979/980 I80M/E135V/I147C + 981/982 L81W/M126Y/A144G/Q188R/S208V/L228M +  983/984 T51I/E189Y + 985/986 A45G/T51I/Q226G +  987/988 T20L +  989/990M126Y/A144G/Q188R/E189V/Q226G +  991/992 H8A/I147C/Q181R/R250A + 993/994 Q236W +  995/996 Q188R +  997/998 Q226G +  999/1000 L228M +1001/1002 L81W + 1003/1004 M126Y + 1005/1006 H240F + 1007/1008 S208V +1009/1010 A231V + 1011/1012 Q236G + 1013/1014 Q236I + 1015/1016 S6T +1017/1018 N230E + 1019/1020 L24V + 1021/1022 A144G + 1023/1024 T51I +1025/1026 I147C + 1027/1028 E189V + 1029/1030 A45T + 1031/1032 E189Y +1033/1034 A36I + 1035/1036 I147M + 1037/1038 N40V + 1039/1040 H8A +1041/1042 S46Q + 1043/1044 M126Q + 1045/1046 R250A + 1047/1048 K43P +1049/1050 E135V + 1051/1052 M126V + 1053/1054 Q181R + 1055/1056 L57S +1057/1058 A22G + 1059/1060 M9V + 1061/1062 K235G + 1063/1064 E142L +1065/1066 Q236A + 1067/1068 A143G + 1069/1070 P41G + 1071/1072 N103G +1073/1074 E3N + 1075/1076 E189T + 1077/1078 A207C + 1079/1080 A19V +1081/1082 L57T + 1083/1084 N40L + 1085/1086 I80M + 1087/1088 A207G +1089/1090 D58T + 1091/1092 A149F + 1093/1094 E232S + 1095/1096 H240W +1097/1098 M126L + 1099/1100 V141L + 1101/1102 I86L + 1103/1104 S146V +1105/1106 E142I + 1107/1108 F134L + “Levels of increased activity weredetermined relative to the reference polypeptide of SEQ ID NO: 776 anddefined as follows: ““+”” 1.02 to 1.18, ““++”” > 1.18, ““+++”” > 1.42”

EXAMPLE 9 Improvements Over SEQ ID NO: 868 Toward the Synthesis of5′-Isobutyryluridine

SEQ ID NO: 868 was selected as the parent enzyme based on the results ofscreening variants toward the synthesis of 5′-isobutyryluridine(compound (1)) from 5′-isobutyryl ribose (compound (4)) and uracil(compound (3))(Scheme III).

Libraries of engineered genes were produced using well-establishedtechniques (e.g., saturation mutagenesis, and recombination ofpreviously identified beneficial mutations). The polypeptides encoded byeach gene were produced in HTP as described in Example 2. For allvariants, the cell pellets were lysed by adding 400 μL lysis buffer(containing 100 mM triethanolamine buffer, pH 7.5, 1 g/L lysozyme, and0.5 g/L PMBS) and shaking at room temperature for 2 hours on a tabletopshaker. The plates were centrifuged at 4,000 rpm for 15 minutes at 4° C.to remove cell debris.

Reactions were performed in a 96-well format in 2 mL deep-well plates,with 100 μL total volume. Reactions included 72.6 g/L (330 mM)5′-isobutyryl ribose solution (19.5 wt %), 37 g/L (330 mM) uracil, 0.76g/L (0.5 mol %) TPP, 0.94 g/L (0.5 mol %) ATP, 0.14 g/L (0.05 mol %)FAD, 46.3 g/L (412 mM) pyruvic acid, 3.7 g/L (33 mM) K₂HPO₄, 0.5 wt %acetate kinase (ACK-101 Codexis, Inc.), 1.9 wt % evolved MTR-kinase (SEQID NO: 1198), 0.5 wt % catalase, 0.8 wt % pyruvate oxidase (SEQ ID NO:1200), and 10 mM MgCl₂. The reactions were set up as follows: (i) anaqueous solution of pyruvic acid and monopotassium phosphate was cooledto 0° C. before addition of 5′-isobutyryl ribose-1-phosphate solution(portion-wise). The resultant solution was pH adjusted to pH 7.2 using8N KOH while maintained at 0° C. To this solution was added: MgCl₂, TPP,FAD, and ATP followed by AcK, MTR-kinase, catalase, and pyruvateoxidase. The resulting solution (90 μL) was then aliquoted into eachwell of the 96-well plates. (ii) 10 μL, of 100× diluted UP lysate wasthen added into the wells to initiate the reaction. The reaction platewas sealed with air-permeable tape and incubated at 25° C. with 250 rpmshaking for 18-20 hat 85% humidity. The reactions were quenched with1000 μL, of a 1:1 mixture of acetonitrile in 20 mM TEoA, pH 7.5 buffer.The quenched reactions were shaken vigorously for 15 min on a tabletopshaker followed by centrifugation at 4,000 rpm for 10 mins at roomtemperature to pellet any precipitate. Supernatant (15 μL) was thentransferred into a 96-well round bottom plate prefilled with 120 μL of1:9 mixture of acetonitrile in 20 mM TEoA, pH 7.5 buffer. The sampleswere analyzed according to the reverse-phase analytical methodsummarized in Table 5.1.

Activity relative to SEQ ID NO: 868 (Activity FIOP) was calculated asthe peak area of 5′-isobutyryluridine product formed by the reactionwith the variant compared with the peak area of 5′-isobutyryluridineproduct produced by the reaction with SEQ ID NO: 868. The results areshown in Table 6.1.

TABLE 6.1 FIOP 5-IBU SEQ ID Relative NO: Amino Acid Differences to SEQID (nt/aa) (Relative to SEQ ID NO: 868) NO: 868 1109/1110N103G/M126Y/V189E/S233D/K235P +++ 1111/1112 S6T/M126Y/S233D/K235P +++1113/1114 L24V/S46Q/I147C/V189E +++ 1115/1116L24V/N103G/M126Y/I147C/Q181R/V189E/S208V/S233D +++ 1117/1118S6T/S46Q/M126Y/I147C/Q181R/K235P/H240F +++ 1119/1120S6T/L24V/M126Y/V189E +++ 1121/1122 L24V/Q181R/V189E/N230E/S233D/K235P+++ 1123/1124 I147C/Q181R/S233D +++ 1125/1126 S6T/L24V/N103G/V189E +++1127/1128 L24V/V189E/N230E ++ 1129/1130 S6T/L24V ++ 1131/1132L24V/N103G/M126Y/I147C ++ 1133/1134 S6T/N103G/V189E/K235P ++ 1135/1136S6T/S46Q/I147C/V189E/H240F ++ 1137/1138 V189E/K235P ++ 1139/1140S6T/L24V/I147C ++ 1141/1142 S6T/M126Y/Q181R/V189E/K235P ++ 1143/1144S6T/N103G/I147C/N230E/S233D ++ 1145/1146 L24V/I147C ++ 1147/1148S6T/L24V/K43P/S46Q/Q181R/V189E ++ 1149/1150 S6T/H8A/M9V/M126Y/I147C ++1151/1152 K43P/S46Q/M126Y/I147C/V189E/H240F ++ 1153/1154S6T/L24V/S46Q/I147C/H240F + 1155/1156L24V/S46Q/S208V/N230E/S233D/K235P + 1157/1158 S6T/M126Y/Q181R/V189E +1159/1160 L24V/S46Q/M126Y + 1161/1162 S208V/S233D + 1163/1164L24V/K43P/S46Q/I147C/N230E/K235P + 1165/1166 M9V/S46Q/I147C/S233D +1167/1168 N103G/I147C/Q181R + 1169/1170S6T/L24V/S46Q/N103G/Q181R/S208V + 1171/1172L24V/I147C/V189E/N230E/S233D + 1173/1174S6T/L24V/K43P/M126Y/I147C/V189E + 1175/1176 S6T/M9V/Q181R/V189E/K235P +1177/1178 V189E + 1179/1180 L24V/S208V + 1181/1182M9V/K43P/S46Q/N103G/V189E/S233D/H240W + 1183/1184S6T/M9V/S208V/S233D/K235P + 1185/1186 K43P/N103G/V189E/S208V/S233D +1187/1188 S6T/M9V/L24V/Q181R/V189E + 1189/1190M9V/S46Q/M126Y/I147C/Q181R + 1191/1192 S6T/N103G + 1193/1194 S6T +1195/1196 S6T/Q181R/N230E/S233D/K235P + “Levels of increased activitywere determined relative to the reference polypeptide of SEQ ID NO: 868and defined as follows: ““+”” 1.06 to 1.22, ““++”” > 1.22, ““+++”” >1.30”

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

We claim:
 1. An engineered uridine phosphorylase comprising apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NOs: 2, 4, 246, 594, 776, and/or 868, or a functional fragmentthereof, wherein the polypeptide sequence of said engineered uridinephosphorylase comprises at least one substitution or substitution setand wherein the amino acid positions of said polypeptide sequence arenumbered with reference to SEQ ID NOs: 2, 4, 246, 594, 776, and/or 868.2. The engineered uridine phosphorylase of claim 1, wherein saidpolypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 2, and wherein the polypeptide sequence of said engineereduridine phosphorylase comprises at least one substitution orsubstitution set at one or more positions in said polypeptide sequenceselected from 6, 7, 9, 14, 14/38/40/146/147/179/235/236,14/38/86/146/147/235/236/240, 14/40, 14/40/86/147/193/236/240,14/40/136/179/236/240, 14/40/146/235/236, 14/40/147/181/193/235,14/40/235, 14/86/146, 14/146/147/181/240, 14/146/236/240,14/147/193/235/236/240, 14/179/181/193/235/240, 14/235/236, 29, 31,38/40/86/146/147/179/181, 38/40/86/147/236/240, 40, 40/43/86/146/240,40/43/86/147/235, 40/43/146/147, 40/43/147/179/236/240,40/43/147/179/240, 40/43/147/236/240, 40/86/146/235/236,40/86/147/235/236/240, 40/86/179/235/240, 40/86/235/236/240,40/146/147/240, 40/147/240, 40/235, 40/235/236/240, 40/236, 40/236/240,42/235/236, 43/86/147/181/240, 43/146/147/235/236/240, 43/146/179/240,43/147, 43/147/179/181, 47, 47/88, 64, 73, 80, 86,86/136/146/147/179/181, 86/136/146/147/179/235/236, 86/147/179/181,86/235, 86/235/236/240, 86/236/240, 86/240, 92, 97, 99, 103, 103/249,104, 105, 106, 110, 146/147, 146/147/235/236, 146/235/240, 146/236/240,146/240, 147/179/181, 147/235/240/249, 157, 167, 179, 179/181,179/181/193/240, 181, 184, 216, 226, 228, 231, 233, 235, 235/236, 236,236/240, 237, 239, 240, and 245, wherein the amino acid positions ofsaid polypeptide sequence are numbered with reference to SEQ ID NO: 2.3. The engineered uridine phosphorylase of claim 1, wherein saidpolypeptide sequence of said engineered uridine phosphorylase has atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to SEQ ID NO: 4, and wherein saidpolypeptide sequence of said engineered uridine phosphorylase comprisesat least one substitution or substitution set at one or more positionsselected from 3, 3/9/216, 3/9/216/236, 3/9/235/237, 3/31/47/179/181/216,3/31/47/179/181/237, 3/31/47/179/216, 3/31/47/179/216/237, 3/31/179,3/31/179/181, 3/31/179/181/237, 3/31/179/216, 3/31/181/216,3/31/181/237, 3/47/179/181/216, 3/47/179/216/237, 3/47/181, 3/179,3/179/181, 3/179/181/216, 3/179/181/237, 3/179/216, 3/179/216/237,3/179/237, 3/181/216, 3/181/216/237, 3/216/236/240, 9/216/236/237,9/237, 13, 24, 31, 31/47, 31/47/179/181/237, 31/47/179/216, 31/47/181,31/47/216, 31/179, 31/179/181, 31/179/216, 31/181, 31/181/216,31/181/216/237, 31/181/237, 31/216, 31/216/237, 31/236/237/240, 31/237,33, 46, 47, 47/147/181/231, 47/179/181, 47/179/181/216, 47/179/184,47/179/216, 47/181/216, 47/181/216/237, 47/181/231, 47/216, 52, 63, 67,83, 87/160, 92, 95, 97, 99, 100, 101, 105, 106, 108, 111, 137, 151, 152,155, 159, 160, 170, 173, 177, 179, 179/181, 179/181/216,179/181/216/237, 179/181/231, 179/181/241, 179/216, 179/228/231,179/237, 181, 181/216, 181/216/237, 181/237, 183, 185, 188, 189, 191,201, 216, 216/237, 218, 222, 228, 231, 233, 235, 235/237, 236,236/237/240, 237, 238, 240, 241, and 248, wherein the amino acidpositions of said polypeptide sequence are numbered with reference toSEQ ID NO:
 4. 4. The engineered uridine phosphorylase of claim 1,wherein said polypeptide sequence of said engineered uridinephosphorylase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:246, and wherein said polypeptide sequence of said engineered uridinephosphorylase comprises at least one substitution or substitution set atone or more positions selected from 3/24/33/47/100/183/185,3/24/33/47/100/216/228/233, 3/24/33/100/183/185/228, 3/24/33/108,3/24/47/100, 3/24/47/100/108/111/160/185/233/241, 3/24/47/108/160/241,3/24/47/160/189, 3/24/47/189/228/233, 3/24/47/228, 3/24/47/228/233,3/24/95/100, 3/24/95/100/160/189/228/241, 3/24/100,3/24/100/160/218/241, 3/24/111, 3/24/111/183/228/233/241,3/24/111/228/233, 3/24/183/185/216, 3/24/189/233, 3/33/47/95/100/241,3/33/47/100/108/189/216/228/233, 3/33/47/100/111/228,3/33/47/100/111/233/241, 3/33/47/100/216, 3/33/47/108/111/233,3/33/47/108/189/233/241, 3/33/160/233, 3/47, 3/47/95/100/108/189/233,3/47/95/100/111/241, 3/47/95/160/189, 3/47/100/108/183/185/189/241,3/47/100/160/185, 3/47/100/185/189/228, 3/47/108/111,3/47/183/189/228/233, 3/47/189, 3/47/228/233, 3/95/100/160/228/233,3/95/100/183/216/228/233, 3/95/100/183/233, 3/95/185/189/216, 3/95/189,3/95/233, 3/160, 3/183/185/189/228/233, 3/183/189/228/233, 3/185,3/185/189, 3/189, 24, 24/33/47, 24/33/47/228/241, 24/33/100/108/241,24/47/95/100, 24/47/95/100/160/228/233/241, 24/47/185/216/218,24/47/216, 24/95/183, 24/100/160/233, 24/160/183/185, 24/189/228/233,33/47/95/100/233, 33/47/95/100/233/241, 33/47/160, 33/47/233,33/100/183/185, 33/100/185/233, 47, 47/100/111/233, 47/100/189,47/100/189/233, 47/108/160/228/241, 47/111, 47/160/185/189/233,47/228/233, 95/100/183, 95/100/189, 95/100/228, 95/100/228/233,95/100/233, 100/160/185, 100/228/233, 108, 108/183/189/233,108/185/216/228/233, 160/233, 228, and 228/233, wherein the amino acidpositions of said polypeptide sequence are numbered with reference toSEQ ID NO:
 246. 5. The engineered uridine phosphorylase of claim 1,wherein said polypeptide sequence of said engineered uridinephosphorylase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:594, and wherein said polypeptide sequence of said engineered uridinephosphorylase comprises at least one substitution or substitution set atone or more positions selected from 6,6/9/29/40/100/121/126/179/181/189/237, 6/9/121/179/181,6/46/52/63/97/121/126/179, 6/52/180/181, 6/63/126/179/242, 9,9/40/46/97/100/106/135/179/181/207/231, 9/52/126/189/242,9/97/100/106/126/180/207/231, 9/181/242, 29, 40, 46, 52, 52/63/126,52/179/189, 61, 63, 97, 100, 106, 121, 126, 126/180/189, 135, 142, 179,180, 181, 189, 201, 207, 230, 231, 236, 237, and 242, wherein the aminoacid positions of said polypeptide sequence are numbered with referenceto SEQ ID NO:
 594. 6. The engineered uridine phosphorylase of claim 1,wherein said polypeptide sequence of said engineered uridinephosphorylase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:776, and wherein said polypeptide sequence of said engineered uridinephosphorylase comprises at least one substitution or substitution set atone or more positions selected from 3, 3/8/22/36/181/235/250, 6,6/45/51/81/126/226/233, 6/45/51/144, 6/45/51/188/189, 6/45/51/189/208,6/45/51/189/228/236, 6/45/51/208/233, 6/45/149/188/189/208/233/236,6/51/126/144/208, 6/51/126/189/208/233/236, 6/51/126/189/231/236,6/51/126/189/233, 6/51/188/189/208/226/228, 6/51/188/189/236,6/51/189/208/231/233, 6/51/208/226/233, 6/51/208/231, 6/126/188/231/233,6/144/208, 6/188/189/208/228/233, 8, 8/36/143/147/235, 8/36/181/235,8/142/147, 8/147/181/250, 8/147/235, 9, 19, 20, 22, 22/147/181/235/250,24, 36, 36/143/147/181, 36/143/147/235, 40, 41, 43, 45, 45/51,45/51/126/144/208/226/228, 45/51/144, 45/51/144/208/226/231/233,45/51/188/189, 45/51/189/233, 45/51/208/226/231, 45/51/208/233,45/51/226, 45/126/189, 45/126/189/208/226, 45/144/189/228,45/144/226/231/233, 45/188/189, 45/188/189/208/228, 45/188/189/226/228,45/188/189/231/233, 45/189, 45/189/208, 46, 51, 51/126/144/208,51/126/144/226/231/233/236, 51/126/208, 51/144, 51/144/226,51/188/189/228, 51/189, 51/189/208/226, 51/208, 51/233, 57, 58, 80,80/135/147, 81, 81/126/144/188/208/228, 86, 103, 126,126/144/188/189/226, 134, 135, 141, 142, 143, 143/147/235, 144,144/188/228, 146, 147, 149, 181, 188, 188/189/233, 189, 207, 208,208/226/233, 208/228, 208/231/233, 226, 228, 230, 231, 232, 233, 235,236, 240, and 250, wherein the amino acid positions of said polypeptidesequence are numbered with reference to SEQ ID NO:
 776. 7. Theengineered uridine phosphorylase of claim 1, wherein said polypeptidesequence of said engineered uridine phosphorylase has at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 868, and wherein said polypeptidesequence of said engineered uridine phosphorylase comprises at least onesubstitution or substitution set at one or more positions selected from6, 6/8/9/126/147, 6/9/24/181/189, 6/9/181/189/235, 6/9/208/233/235,6/24, 6/24/43/46/181/189, 6/24/43/126/147/189, 6/24/46/103/181/208,6/24/46/147/240, 6/24/103/189, 6/24/126/189, 6/24/147,6/46/126/147/181/235/240, 6/46/147/189/240, 6/103, 6/103/147/230/233,6/103/189/235, 6/126/181/189, 6/126/181/189/235, 6/126/233/235,6/181/230/233/235, 9/43/46/103/189/233/240, 9/46/126/147/181,9/46/147/233, 24/43/46/147/230/235, 24/46/126, 24/46/147/189,24/46/208/230/233/235, 24/103/126/147, 24/103/126/147/181/189/208/233,24/147, 24/147/189/230/233, 24/181/189/230/233/235, 24/189/230, 24/208,43/46/126/147/189/240, 43/103/189/208/233, 103/126/189/233/235,103/147/181, 147/181/233, 189, 189/235, and 208/233, wherein the aminoacid positions of said polypeptide sequence are numbered with referenceto SEQ ID NO:
 868. 8. The engineered uridine phosphorylase of claim 1,wherein said engineered uridine phosphorylase comprises a polypeptidesequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of atleast one engineered uridine phosphorylase variant set forth in 1.2,2.2, 3.1, 4.1, 5.2, and/or 6.1.
 9. The engineered uridine phosphorylaseof claim 1, wherein said engineered uridine phosphorylase comprises apolypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NOs:2, 4, 246, 594, 776, and/or
 868. 10. The engineered uridinephosphorylase of claim 1, wherein said engineered uridine phosphorylasecomprises a variant engineered uridine phosphorylase set forth in SEQ IDNOs: 4, 246, 594, 776, and/or
 868. 11. The engineered uridinephosphorylase of claim 1, wherein said engineered uridine phosphorylasecomprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the sequence of at least one engineered uridine phosphorylase variantset forth in the even numbered sequences of SEQ ID NOs: 4-1196
 12. Theengineered uridine phosphorylase of claim 1, wherein said engineereduridine phosphorylase comprises a polypeptide sequence forth in at leastone of the even numbered sequences of SEQ ID NOs: 4-1196.
 13. Theengineered uridine phosphorylase of claim 1, wherein said engineereduridine phosphorylase comprises at least one improved property comparedto wild-type Escherichia coli uridine phosphorylase.
 14. The engineereduridine phosphorylase of claim 13, wherein said improved propertycomprises improved activity on one or more substrates.
 15. Theengineered uridine phosphorylase of claim 14, wherein said one or moresubstrates comprises 5′-isobutyryl ribose-1-phosphate (compound (2))and/or uracil (compound (3)).
 16. The engineered uridine phosphorylaseof claim 13, wherein said improved property comprises improvedproduction of 5′-isobutyryluridine, compound (1).
 17. The engineereduridine phosphorylase of claim 1, wherein said engineered uridinephosphorylase is purified.
 18. The engineered uridine phosphorylase ofclaim 1, wherein said engineered uridine phosphorylase is part of amulti-enzyme system for producing a nucleoside analogue.
 19. Acomposition comprising at least one engineered uridine phosphorylase ofclaim
 1. 20. A polynucleotide sequence encoding at least one engineereduridine phosphorylase of claim
 1. 21. A polynucleotide sequence encodingat least one engineered uridine phosphorylase, said polynucleotidesequence comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NOs:1, 3, 245, 593, 775 and/or 867, wherein the polynucleotide sequence ofsaid engineered uridine phosphorylase comprises at least onesubstitution at one or more positions.
 22. A polynucleotide sequenceencoding at least one engineered uridine phosphorylase comprising atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to SEQ ID NOs: 1, 3, 245, 593, 775and/or 867, or a functional fragment thereof.
 23. The polynucleotidesequence of claim 20, wherein said polynucleotide sequence is operablylinked to a control sequence.
 24. The polynucleotide sequence of claim20, wherein said polynucleotide sequence is codon optimized
 25. Thepolynucleotide sequence of claim 20, wherein said polynucleotidesequence comprises a polynucleotide sequence set forth in the oddnumbered sequences of SEQ ID NOs: 3-1195.
 26. An expression vectorcomprising at least one polynucleotide sequence of claim
 20. 27. A hostcell comprising at least one expression vector of claim
 26. 28. A hostcell comprising at least one polynucleotide sequence of claim
 20. 29. Amethod of producing an engineered uridine phosphorylase in a host cell,comprising culturing the host cell of claim 27, under suitableconditions, such that at least one engineered uridine phosphorylase isproduced.
 30. The method of claim 29, further comprising recovering atleast one engineered uridine phosphorylase from the culture and/or hostcell.
 31. The method of claim 29, further comprising the step ofpurifying said at least one engineered uridine phosphorylase.